Self-contained biological assay apparatus, methods, and applications

ABSTRACT

A self-contained, fully automated, biological assay-performing apparatus includes a housing; a dispensing platform including a controllably-movable reagent dispensing system, disposed in the housing; a reagent supply component disposed in the housing; a pneumatic manifold removably disposed in the housing in a space shared by the dispensing platform, removably coupled to a fluidic transport layer and a plurality of reservoirs, wherein the fluidic transport layer, the reservoirs, and a test sample to be introduced therein are disposed in the housing in the space separate from the dispensing platform; a pneumatic supply system removably coupled to the pneumatic manifold in the housing in a space separate from the dispensing platform; and a control system coupled to at least one of the dispensing platform and the pneumatic supply system, disposed in the housing.

CROSS-REFERENCE TO RELATED APPLICATIONS

The instant application is a continuation of U.S. application Ser. No.13/836,032, filed Mar. 15, 2013, now U.S. Pat. No. 8,986,614, which is acontinuation-in-part (CIP) application of U.S. application Ser. No.13/033,165 filed on Feb. 23, 2011, now U.S. Pat. No. 8,383,039 andclaims priority thereto an to the following U.S. provisionalApplications: Ser. No. 61/444,952 filed on Feb. 21, 2011; Ser. No.61/445,125 filed on Feb. 22, 2011; Ser. No. 61/445,130 filed on Feb. 23,2011; Ser. No. 61/346,202 filed on May 19, 2010; Ser. No. 61/355,773filed on Jun. 17, 2010; Ser. No. 61/405,339 filed Oct. 21, 2010; Ser.No. 61/307,186, filed on Feb. 23, 2010; Ser. No. 61/307,121 filed onFeb. 23, 2010; Ser. No. 61/393,237 filed on Oct. 14, 2010; 61/374,302filed on Aug. 17, 2010, the subject matter of all of which areincorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Embodiments of the present invention relate generally to the field ofmicrofluidics and, more particularly to a self-contained,microfluidic-based biological assay apparatus and associated methods andapplications.

2. Technical Background

Biochemical assays are generally used in research, clinical,environmental and industrial settings to detect or quantify the presenceor amount of certain gene sequences, antigens, diseases, and pathogens.The assays are often used to identify organisms including parasites,fungi, bacteria and viruses present in a host organism or a sample.Under certain conditions assays may provide a measure of quantificationwhich may be used to calculate the extent of infection or disease and tomonitor the state of a disease over time. In general, biochemical assayseither detect antigens (immunoassays) or nucleic acids (nucleicacid-based or molecular assays) extracted from samples derived fromresearch, clinical, environmental or industrial sources.

Molecular biology, which includes nucleic acid-based assays, can bebroadly defined as the branch of biology that deals with the formation,structure and function of macromolecules such as nucleic acids andproteins and their role in cell replication and the transmission ofgenetic information, as well as the manipulation of nucleic acids, sothat they can be sequenced, mutated, and further manipulated into thegenome of an organism to study the biological effects of the mutation.

The conventional practice of biochemistry and molecular biology canrequire physical process resources on a scale that are frequentlyinversely proportional to the size of the subject being studied. Forexample, the apparatus and process chemistry associated with thepreparation and purification of a biological sample such as a nucleicacid fragment for prospective analysis may easily require a full scalebio-laboratory with sterile facilities. Furthermore, an environmentallyisolated facility of similar scale may typically be required to carryout the known nucleic acid amplification procedures such as polymerasechain reaction (PCR) for amplifying the nucleic acid fragment.

“Microfluidics” generally refers to systems, devices, and methods forprocessing small volumes of fluids. Microfluidic systems can integrate awide variety of operations for manipulating fluids. Such fluids mayinclude chemical or biological samples. These systems also have manyapplication areas, such as biological assays (for, e.g., medicaldiagnoses, drug discovery and drug delivery), biochemical sensors, orlife science research in general as well as environmental analysis,industrial process monitoring and food safety testing.

One type of microfluidic device is a microfluidic chip. Microfluidicchips may include micro-scale features (or “microfeatures”), such aschannels, valves, pumps, reactors and/or reservoirs for storing fluids,for routing fluids to and from various locations on the chip, and/or forreacting reagents. However, existing microfluidic systems lack adequatemechanisms for allowing controlled manipulation of multiple fluidsexcept via prescribed flow patterns, hence limiting the practicalitywith which the systems can be utilized in various chemical or biologicalassays. This is because real-world assays often require repetitivemanipulation of different reagents for various analytical purposes.

Moreover, many existing microfluidic devices are restricted for onespecific use and cannot be easily adapted or customized for otherapplications without being completely redesigned. These devices lackmodularity, and therefore cannot share common device components thatallow one design to perform multiple functions. This lack of flexibilityleads to increased production costs as each use requires the productionof a different system.

Furthermore, many existing microfluidic systems lack any means forstraightforward end-point assays that are able to easily detectinteractions or existence of analytes resulting from the assays. By wayof example, visual detection of sample color changes after an assay isoften used to evaluate the assay results.

Thus there exists a need for improved microfluidic systems forprocessing fluids for analysis of biological or chemical samples, and inparticular, in the detection and analysis of biologically activemacromolecules derived from such samples such as DNA, RNA, amino acidsand proteins. It is desired that the systems are mass producible,inexpensive, and preferably disposable. It is desired that the systemsbe simple to operate and that many or substantially all of the fluidprocessing steps be automated. It is desired that the systems becustomizable, and be modular such that the system can be easily andrapidly reconfigured to suit various applications in which the detectionof macromolecules is desired. It is also desired that the systems beable to provide straightforward and meaningful assay results.

When performing a nucleic acid-based assay, preparation of the sample isthe first and most critical step to release and stabilize target nucleicacids that may be present in the sample. Sample preparation can alsoserve to eliminate nuclease activity and remove or inactivate potentialinhibitors of nucleic acid amplification or detection of the targetnucleic acids. The method of sample preparation can vary and will dependin part on the nature of the sample being processed. Various lysisprocedures are well known in the art and are designed to specificallyisolate nucleic acids from cells or viruses suspended in the originalsample.

Following lysis, the released nucleic acids in the sample need to bepurified so that the potential inhibitors for the amplification reactionare removed from the nucleic acids. Generally, purification is acumbersome and repetitive set of tasks consuming large amounts ofreagents, capital equipment, and labor and it is often the step mostassociated with failure of down-steam amplification reactions.

Following purification it is generally desirable to amplify specificnucleic acid sequences using any of several nucleic acid amplificationprocedures which are well known in the art. Specifically, nucleic acidamplification is the enzymatic synthesis of nucleic acid amplicons(copies) which contain a sequence that is homologous to a nucleic acidsequence being amplified. Examples of nucleic acid amplificationprocedures practiced in the art include the polymerase chain reaction(PCR), strand displacement amplification (SDA), ligase chain reaction(LCR), Nucleic Acid Sequence Based Amplification (NASBA),transcription-associated amplification (TAA), Cold PCR, andNon-Enzymatic Amplification Technology (NEAT). Nucleic acidamplification is especially beneficial when the amount of targetsequence present in a sample is very low. By amplifying the targetsequences and detecting the amplicon synthesized, the sensitivity of anassay can be vastly improved, since fewer target sequences are needed atthe beginning of the assay to better ensure detection of nucleic acid inthe sample belonging to the organism or virus of interest.

Detection of a targeted nucleic acid sequence requires the use of anucleic acid probe having a nucleotide base sequence that issubstantially complementary to the targeted sequence or, alternatively,its amplicon. Under selective assay conditions, the probe will hybridizeto the targeted sequence or its amplicon in a manner permitting apractitioner to detect the presence of the targeted sequence in asample. Effective probes are designed to prevent nonspecifichybridization with any nucleic acid sequence that will interfere withdetecting the presence of the targeted sequence. Probes and/or theamplicons may include a label capable of detection, where the label is,for example, a radiolabel, fluorescent dye, biotin, enzyme,electrochemical or chemiluminescent compound.

When performed manually, the complexity and shear number of processingsteps associated with a nucleic acid-based assay introduce opportunitiesfor practitioner-error, exposure to pathogens, and cross-contaminationbetween assays, and others. Following a manual format, the practitionermust safely and conveniently juxtapose the test samples, reagents, wastecontainers, assay receptacles, pipette tips, aspirator device, dispenserdevice, while being especially careful not to confuse racks, testsamples, assay receptacles, and associated tips, or to knock over anytubes, tips, containers, or instruments. In addition, the practitionermust carefully perform aspirating and dispensing steps with handheld,non-fixed instruments in a manner requiring precise execution to avoidundesirable contact between assay receptacles, aerosol formation, oraspiration of magnetic particles or other substrates used in atarget-capture assay.

A need exists for an automated analyzer that addresses many of theconcerns associated with manual approaches to performing nucleicacid-based assays. In particular, significant advantages can be realizedby automating the various process steps of a nucleic acid-based assay,including greatly reducing the risk of user-error, pathogen exposure,contamination and spillage. Automating the steps of a nucleic acid-basedassay will also reduce the amount training required for practitionersand virtually eliminate sources of physical injury attributable tohigh-volume manual applications.

Real-time polymerase chain reaction (“PCR”), also known as quantitativereal time polymerase chain reaction (“qrt-PCR”) among otherdesignations, is a molecular biology tool used to simultaneously amplifya target DNA molecule using the well-known PCR process while quantifyingthe target DNA either as an absolute amount or a relative amountcompared to another input. With standard PCR, the product is detectedfollowing completion of the reaction. To perform qrt-PCR, in contrast,the user amplifies the target DNA molecule much the same way, butdetects the targeted DNA molecule in real time as the polymerase chainreaction progresses. Some of the most common methods used to detect theqrt-PCR product in real time are to utilize a non-specific fluorescentdye that incorporates into a double-stranded DNA product, or to use asequence specific probe labeled with a fluorescent reporter that willonly fluoresce when the probe hybridizes with the target sequence. Ifthese methods are employed, for example, additional equipment such as alight source and a fluorescence detector will be required.

To quantify the qrt-PCR product, the detected fluorescence is plotted ona logarithmic scale against the cycle number. The amount of target inthe pending reaction can then be determined by comparing theexperimental results to standard results obtained using known amounts ofproduct. This is, however, just one of the ways that the qrt-PCR productcan be quantified.

Quantitative real-time PCR has numerous applications, particularly inthe study of molecular biology. One particular use of qrt-PCR is toobtain quantitative information about pathogens in a sample. Forquantitation a real-time measurement of fluorescent intensity, orreal-time measurement of another parameter indicating an increasedconcentration of amplicons during an amplification reaction, isnecessary. To differentiate multiple targets, particular primers withspecific probes must be designed for each target. Or, in a lessdesirable case, only the primers need be designed and a dye such as SYBRGreen applied to the reaction to indicate growing concentration ofamplicons. For example, if there are 10 potential targets then typically10 different probes are needed. The current state-of-the-art fordetector/probe combinations allows for multiplexing of up toapproximately 4 or 5 targets simultaneously. Most real-time PCR systems,however, are equipped only with two detectors for multiplexingdetection. Using such a system, 6 separate PCR reactions are necessaryto differentiate 6 hepatitis C virus (“HCV”) genotypes

SUMMARY OF THE INVENTION

Embodiments and aspects of the present invention address the needsdescribed above by providing a self-contained, microfluidic-basedbiological assay apparatus, associated methods, and applicationsthereof.

According to a non-limiting, exemplary embodiment, a self-contained,fully automated, biological assay-performing apparatus includes ahousing; a dispensing platform including a controllably-movable reagentdispensing system, disposed in the housing; a reagent supply componentdisposed in the housing; a pneumatic manifold removably disposed in thehousing in a space shared by the dispensing platform, removably coupledto a fluidic transport layer and a plurality of reservoirs, wherein thefluidic transport layer, the reservoirs, and a test sample to beintroduced therein are disposed in the housing in the space separatefrom the dispensing platform; a pneumatic supply system removablycoupled to the pneumatic manifold in the housing in a space separatefrom the dispensing platform; and a control system coupled to at leastone of the dispensing platform and the pneumatic supply system, disposedin the housing.

According to a non-limiting aspect, the dispensing platform furtherincludes a motion control system operatively coupled to the reagentdispensing system, wherein the reagent dispensing system includes areagent dispenser component having a distal dispensing end; and a cameraconnected to the reagent dispensing system having a field of view thatincludes at least a selected region of interest of the reservoirs.

According to a non-limiting aspect, the pneumatic manifold is interfacedwith a microfluidic system having at least one assay capacity. Themicrofluidic system may further comprise a multi-layer, monolithic,polymeric, non-elastomeric microfluidic component having a givenconfiguration of microfeatures including a plurality ofpneumatically-activated diaphragms. The pneumatic manifold may have oneor more pneumatic only (i.e., absence of ‘fluidic’) ports on anunderside thereof, and one or more pneumatic only channels disposedtherein in fluid connection with one or more valves in the fluidictransport layer and the one or more pneumatic only ports, wherein theone or more pneumatic only ports have a fixed configuration, and the oneor more pneumatic only channels have a given configuration specificallycorresponding to a given configuration of the one or morepneumatically-activated diaphragms in the fluidic transport layer. Thepneumatic supply system may further include one or more aperture tubesthat provide a passage of the pneumatic signal there through, in fluidconnection with the one or more pneumatic only ports, wherein the one ormore aperture tubes have a fixed configuration specificallycorresponding to the fixed configuration of the one or more pneumaticonly ports of the pneumatic manifold, removably connected to thepneumatic manifold. Each multi-layer, monolithic microfluidic componentmay further include a polymeric, non-elastomeric substrate having one ormore fluid channels disposed therein, each of the fluid channels havingan inlet end and an outlet end; at least one reagent reservoir of a typecapable of holding a reagent material; at least one bi-directionaldiaphragm pump comprising at least three non-elastomeric membrane-baseddiaphragm valve structures; and a valve disposed in fluid coupling withthe at least one reagent reservoir and at least one of the inlet ends,wherein the valve is adapted to controllably direct a flow of thematerial from the at least one reagent reservoir to one or morereservoirs via at least one of the channels coupled to the valve,further wherein each multi-layer, monolithic microfluidic componentconsists of a non-elastomeric, polymeric material. Each substrate mayfurther include one or more analysis reservoirs, each analysis reservoirincluding an analysis system disposed therein. The analysis system maybe one of colorimetric, fluorescent colorimetric, chemiluminescent,electrochemical, electrophoretic, lateral flow, protein microarray,nucleic acid microarray, or fluorescent. The apparatus may furtherinclude a securing-ring structure having one or more indentations orchannels in the perimeter thereof, wherein the analysis membrane isoperatively engaged with the securing-ring structure. The securing-ringstructure may comprise two opposing ring structures each having one ormore perimeter indentations, further wherein the analysis membrane isdisposed intermediate the two opposing ring structures. The apparatusmay further include one or more heaters disposed in different locationsto effect heating of the test sample or portions thereof in the analysisprocess. A heater may be disposed in proximity to amagnetically-engageable reservoir. The apparatus may further include atube mounting layer attached to a bottom surface of the substrateincluding one or more tubes each having a proximal end that is fluidlyconnected to a respective fluidic channel and a distal end projectingdownwardly perpendicularly from the substrate; and one or morerespective amplification/reaction chambers attached at a top regionthereof to the tube mounting layer such that the distal end of each tubeis disposed substantially near a bottom region thereof. The apparatusmay further include a magnetic assembly operably disposed under areservoir or channel, wherein the magnetic assembly further comprises amagnet, a magnet holder, a piston rod, and a pneumatic piston assembly(alternatively the piston assembly may be motor or electromagneticallyactivated). A heater assembly may be operably connected to the magneticassembly.

It is therefore a principal object and advantage of the invention toprovide a method for the quantitative detection of one or more targets.

It is another object and advantage of the invention to combinequantitation with multiplexed detection of one or more targets.

It is yet another object and advantage of the invention to combine thequantitative capabilities of real-time nucleic acid amplificationsystems with the multiplexing capabilities of hybridization systems.

Other objects and advantages of the invention will in part be obvious,and in part appear hereinafter.

In accordance with the foregoing objects and advantages, the presentinvention provides a method for detecting in a sample at least one of aplurality of target nucleic acid molecules. The method comprises thesteps of: (i) identifying a first region of each of the plurality oftarget nucleic acid molecules, where the first region is conserved amongthe plurality of target nucleic acid molecules; (ii) producing a firstamplification product of the first region of each target nucleic acidmolecule present in the sample; (iii) detecting the production of thefirst amplification product in real time during the amplification; (iv)identifying a second region of each of the plurality of target nucleicacid molecules, where the second region is not conserved among theplurality of target nucleic acid molecules; (v) producing a secondamplification product of the second region of each target nucleic acidmolecule present in the sample; and (v) detecting the secondamplification product to indicate the presence of each target nucleicacid molecule present in the sample. According to another aspect, thefirst amplification product is produced in the presence of a firstnucleic acid probe that hybridizes to the first region, and the step ofdetecting the amplification product in real time during theamplification comprises detecting hybridization of the first nucleicacid probe to the first region. According to another aspect, the step ofdetecting the second amplification product comprises hybridizing thesecond amplification product to a complementary probe, where the probecan be fixed to a substrate such as a DNA microarray. The method canfurther comprise the steps of: (i) identifying a third region of asecond plurality of target nucleic acid molecules, where the thirdregion is conserved among the second plurality of target nucleic acidmolecules; (ii) producing a third amplification product by amplificationof the third region of each of the second plurality of target nucleicacid molecules present in the sample; and (iii) detecting the productionof the third amplification product in real time. The first amplificationproduct and said third amplification product can be produced in the samereaction.

According to a second aspect is the above method, further comprising thestep of performing quantitative multiplexed detection of each of theplurality of target nucleic acid molecules present in the sample byanalyzing both the quantified amount of target nucleic acid moleculespresent in the sample and the detected presence of each target nucleicacid molecule present in the sample.

According to a third aspect, the presence of one or more target nucleicacid molecules in the sample indicates the presence of a pathogen inthat sample. The method can be capable to detecting, quantifying, and/oridentifying at least two targets—such as pathogens, genes, SNPs,specific nucleic acid sequence, etc.—in the sample.

According to a fourth aspect, the step of identifying a first region ofeach of the plurality of target nucleic acid molecules comprises thesteps of: (i) performing a sequence alignment of at least a segment ofthe nucleic acid sequence of each of the plurality of target nucleicacid molecules; (ii) identifying the first region based on the sequencealignment; (iii) designing a first primer and a second primer that willamplify the first region during the PCR amplification; and (iv)designing a probe that will hybridize to the first region during the PCRamplification of the region. The probe can be designed to have a meltingtemperature that is at least 6° C. higher than the melting temperatureof the first primer and the melting temperature of the second primer.

According to a fifth aspect, the step of identifying the second regionof each of the plurality of target nucleic acid molecules comprises thesteps of: (i) performing a sequence alignment of at least a segment ofthe nucleic acid sequence of each of the plurality of target nucleicacid molecules; (ii) identifying the second region based on the sequencealignment; and (iii) designing a first primer and a second primer thatwill amplify the second region during the PCR amplification. The methodcan also comprise the step of designing a complementary probe that willhybridize to the second region of each of the plurality of targetnucleic acid molecules.

According to a sixth aspect is provided a system for detecting at leastone of a plurality of target nucleic acid molecules. The systemcomprises: (i) a sample comprising at least one of the plurality oftarget nucleic acid molecules; (ii) a first primer pair, the firstprimer pair capable of amplifying a first region of each of theplurality of target nucleic acid molecules to produce a firstamplification product, wherein the first region is conserved among theplurality of target nucleic acid molecules; (iii) a real-time PolymeraseChain Reaction (“PCR”) instrument, wherein the PCR instrument is capableof producing the first amplification product using the first primer pairand is further capable of detecting the first amplification product inreal time; (iv) a second primer pair, the second primer pair capable ofamplifying a second region of each of the plurality of target nucleicacid molecules to produce a second amplification product, wherein thesecond region is not conserved among the plurality of target nucleicacid molecules; (v) a detection device for detecting the secondamplification product. According to an embodiment, the system furthercomprises one or more of the following: a first nucleic acid probe thathybridizes to the first region; and a second nucleic acid probe thathybridizes to the second region, where the second nucleic acid probe canbe fixed to a substrate such as a microarray.

According to a seventh aspect, the system can further comprise one ormore of the following: (i) a third primer pair, the third primer paircapable of amplifying a third region of each of a second plurality oftarget nucleic acid molecules to produce a third amplification product,wherein the third region is conserved among the second plurality oftarget nucleic acid molecules; and (ii) means to purify nucleic acidfrom the sample.

According to an eighth aspect, the system is a kit for detecting in thesample at least one of a plurality of target nucleic acid molecules.

According to a ninth aspect is provided a kit for detecting at least oneof a plurality of target nucleic acid molecules, the kit comprising: (i)a first primer pair, the first primer pair capable of amplifying a firstregion of each of the plurality of target nucleic acid molecules toproduce a first amplification product, wherein the first region isconserved among the plurality of target nucleic acid molecules; (ii) afirst nucleic acid probe that hybridizes to the first region; (iii) asecond primer pair, the second primer pair capable of amplifying asecond region of each of the plurality of target nucleic acid moleculesto produce a second amplification product, wherein the second region isnot conserved among said plurality of target nucleic acid molecules; and(iv) a second nucleic acid probe that hybridizes to the second region.

According to a tenth aspect, the kit can further comprise one or more ofthe following: (i) a microarray; and (ii) means to purify nucleic acidfrom the sample. According to an eleventh aspect, the kit furthercomprises a third primer pair, where the third primer pair is capable ofamplifying a third region of each of a second plurality of targetnucleic acid molecules to produce a third amplification product, thethird region being conserved among the second plurality of targetnucleic acid molecules

Another non-limiting, exemplary embodiment of the invention is directedto an automated process for isolating, amplifying, and analyzing atarget nucleic acid sequence that may be present in a fluid test sample.The process includes the steps of providing a pneumatic manifold thatoperates a microfluidic system having a fluidic transport layer and afluidic channel disposed therein, and reservoirs attached thereto;introducing the fluid test sample into the fluidic channel; providing atleast one reagent to the channel from at least one respective reservoirthat is in fluid connection with the fluidic transport layer; combiningthe fluid test sample and the at least one reagent in a region of thefluidic transport layer, reservoir or amplification reactor;transporting the fluid test sample to a temperature-controlledamplification/reaction reactor that is in operative communication withthe fluidic transport layer; incubating the fluid test sample in theamplification/reaction reactor under conditions sufficient to permit thetarget nucleic acid sequence to be amplified; transporting the fluidtest sample to an analysis reservoir; and analyzing the amplified targetnucleic acid sequence from the test sample, wherein the test sample istransported from a starting location in the fluidic transport layer tothe analysis reservoir separately from any other samples and separatelyfrom the pneumatic manifold and the dispensing system.

In an aspect, the transporting and combining steps are accomplished bypumping the fluid test sample and the volume of at least one reagentthrough the fluidic transport layer via at least one multi-valvediaphragm pump that is operated by the pneumatic manifold. According toan aspect, the analyzing step may further include performing amicroarray analysis in a microarray analysis reservoir in themicrofluidic system. The automated process may further involve providinga microarray analysis membrane in the microarray analysis reservoir;flowing a fluid over a top surface of the microarray analysis membrane;and removing the fluid substantially through a fluid exit route along aperiphery of the microarray analysis membrane. The automated process mayfurther involve providing a microarray analysis membrane in themicroarray analysis reservoir; and, flowing a fluid alternatively backand forth over a top surface of the microarray analysis membrane. Theautomated process may further involve providing heat to the of themicroarray analysis membrane.

In another non-limiting embodiment of the automated process forisolating, amplifying, and analyzing a target nucleic acid sequence thatmay be present in a fluid test sample, the target nucleic acid sequenceis associated with a disease or disorder of interest, an infectiousagent, a pathogen, a predisposition for cancer, or a predisposition forsensitivity to a drug, pharmaceutical composition, chemical or compoundof interest.

In another non-limiting embodiment of the automated process forisolating, amplifying, and analyzing a target nucleic acid sequence thatmay be present in a fluid test sample, the target nucleic acid sequencecomprises a SNP.

In another non-limiting embodiment of the automated process forisolating, amplifying, and analyzing a target nucleic acid sequence thatmay be present in a fluid test sample, the disease or disorder is HPV orsepsis.

In another non-limiting embodiment of the automated process forisolating, amplifying, and analyzing a target nucleic acid sequence thatmay be present in a fluid test sample, the target nucleic acid sequenceis associated with predisposition for warfarin sensitivity orpredisposition for anticoagulation in response to warfarin treatment.

In another non-limiting embodiment of the automated process forisolating, amplifying, and analyzing a target nucleic acid sequence thatmay be present in a fluid test sample, the analyzing step comprisesdetecting an interaction between the amplified target nucleic acidsequence and a probe for the target nucleic acid sequence.

In another non-limiting embodiment of the automated process forisolating, amplifying, and analyzing a target nucleic acid sequence thatmay be present in a fluid test sample, the analyzing step comprisesdetermining presence of, or predisposition for: the disease or disorderof interest, the infectious agent, the pathogen, cancer, or sensitivityto the drug, pharmaceutical composition, chemical or compound ofinterest.

In another non-limiting embodiment of the automated process forisolating, amplifying, and analyzing a target nucleic acid sequence thatmay be present in a fluid test sample, wherein the analyzing stepcomprises determining an amount or level of the amplified target nucleicacid sequence and wherein the method further comprises comparing theamount or level with a preselected amount or level of the target nucleicacid sequence.

The method wherein a difference between the amount or level and thepreselected amount or level is indicative of presence of, orpredisposition for: a disease or disorder of interest, an infectiousagent, a pathogen, cancer, or sensitivity to a drug, pharmaceuticalcomposition, chemical or compound of interest.

These, as well as additional features and advantages of the inventionwill be set forth in the detailed description that follows and will bereadily apparent to those skilled in the art from that description or,recognized by practicing the invention as described in the detaileddescription, the drawing figures, and the appended claims.

It is to be understood that both the foregoing general description andthe following detailed description are merely exemplary of theembodiments of the invention and are intended to provide an overview orframework for understanding the nature and character of the invention asit is claimed. The accompanying drawings are included to provide afurther understanding of the various claimed embodiments and aspects ofthe invention, and are incorporated in and constitute a part of thisspecification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a perspective view of a self-contained biological assayapparatus, according to an exemplary embodiment of the invention;

FIG. 2 shows a front elevational view of the self-contained biologicalassay apparatus shown in FIG. 1;

FIG. 3 shows a top plan view of the self-contained biological assayapparatus shown in FIG. 1;

FIG. 4 shows a perspective view of the self-contained biological assayapparatus shown in FIG. 1;

FIG. 5 shows a top plan view of a “six by four” arrangement ofreservoirs, according to an exemplary aspect of the invention;

FIG. 6 shows a perspective view of a “six by four” arrangement ofreservoirs attached to fluidic transport layers and mounted on thepneumatic manifold, according to an exemplary aspect of the invention;

FIG. 7 shows a cross sectional top plan view of a “six by four”arrangement of the top gasket layer of the pneumatic manifold and theheaters that engage the amplification reactors, according to anexemplary aspect of the invention;

FIGS. 8A-C show, respectively, a top perspective view, a top plan view,and a bottom perspective view of a “six by four” arrangement of thebottom portion of the pneumatic manifold of the self-containedbiological assay apparatus, according to an exemplary aspect of theinvention;

FIG. 9 shows a layered, cross sectional top plan view of a “four assayunit” arrangement of the reservoirs, fluidic channels and amplificationreactors, which are a combination of the fluidic transport layer and thereservoir layer, according to an exemplary aspect of the invention;

FIGS. 10A-K show layered, cross sectional top plan views of exemplaryprocess stages using the channels, reactors, and reservoirs in thecombined fluidic transport layer and the reservoir layer when operatedby the pneumatic manifold, according to illustrative aspects of theinvention;

FIG. 11 shows a perspective view of a “four assay unit” componentincluding the reservoir layer, the fluidic transport layer including afilm layer, and an amplification reactor, according to an exemplaryaspect of the invention;

FIG. 12 shows a cross sectional side view of the “four assay unit”component shown in FIG. 11, according to an exemplary aspect of theinvention;

FIG. 13A shows an exploded view of an exemplary “four assay unit”component including the reservoir layer, the fluidic layer, the filmlayer, the amplification reactors including lumens which are used tofill and empty the amplification reactors, a perforated ring andmembrane system for the analysis reservoir, a silica filter and aperforated retaining ring, according to an exemplary aspect of theinvention; FIG. 13B shows an exemplary perforated ring with its flowchannels, an analysis membrane and a membrane support structure;according to an exemplary aspect of the invention;

FIG. 14A shows a perspective view of an alternative embodiment of thereservoir layer for the analysis area of a “four assay unit” componentincluding a covered and vented component for the analysis reservoirsthat does not employ the perforated ring assembly to hold the analysismembrane, according to an exemplary aspect of the invention; FIG. 14Bshows a close up perspective view of the covered and vented analysisreservoir feature including a model of the analysis membrane and thestep feature used to retain the analysis membrane, according to anexemplary aspect of the invention; FIG. 14C shows a cross sectional topplan view of a four assay unit arrangement of the top gasket layer ofthe pneumatic manifold, the heaters that engage the amplificationreactors and the heaters that engage the under surface of the analysisreservoirs, according to an exemplary aspect of the invention; FIG. 14Dshows the internal pneumatic channels of the upper layers of thepneumatic manifold showing how the pneumatic signals introduced into thebottom layer of the pneumatic manifold from the pneumatic supply systemare further split and addressed to particular gasket layer voids on thesurface of the pneumatic manifold;

FIG. 15A shows an exploded perspective view of a single assay unit of apneumatic manifold, incorporating pneumatically-activated magnets and amultipurpose heater, as well as an optional diaphragm layer for theinterface between the fluidic layer and the pneumatic manifold,according to an exemplary aspect of the invention; FIG. 15B shows anexploded perspective view of the single assay unit shown in FIG. 15Aincluding a thin film layer of the fluidic transport layer and a gasketlayer on the pneumatic manifold, according to an exemplary aspect of theinvention;

FIG. 16 shows a layered, cross sectional top plan view of a “singleassay unit” arrangement of the reservoirs, fluidic channels andamplification reactor, which are a combination of the fluidic transportlayer and the reservoir layer, according to an exemplary aspect of theinvention;

FIGS. 17A-O show layered, top views of exemplary process stages usingthe channels, reactors, and reservoirs in the combined fluidic transportlayer and the reservoir layer when operated by the pneumatic manifold,according to illustrative aspects of the invention;

FIG. 18 shows a cross sectional side view of the pneumatic manifold andthe microfluidic system of a single assay unit showing functionalfeatures of the manifold incorporating pneumatically activated magnetsincluding a non-heated magnetic assembly and multiple heated reactionsites, according to an exemplary aspect of the invention;

FIG. 19 shows a cross sectional side view of the pneumatic manifold andthe microfluidic system of a single assay unit including functionalfeatures of the manifold incorporating pneumatically activated, heatedmagnet assembly and multiple heated reaction sites, according to anexemplary aspect of the invention;

FIG. 20 shows a layered, cross sectional top plan view of a “doubleassay unit” arrangement of the reservoirs, fluidic channels andamplification reactors, which are a combination of the fluidic transportlayer and the reservoir layer, according to an exemplary aspect of theinvention;

FIGS. 21A-K show layered, top views of exemplary process stages usingthe channels, reactors, and reservoirs in the combined fluidic transportlayer and the reservoir layer when operated by the pneumatic manifold,according to illustrative aspects of the invention;

FIG. 22 shows an exploded view of a double assay unit arrangement, whichshows the reservoir layer, the fluidic layer, the film layer, and theamplification reactors including the lumens used to fill and empty theamplification reactors, according to an exemplary aspect of theinvention;

FIG. 23 is an exploded view of a multipurpose heater according to anon-limiting, illustrative aspect of the invention;

FIG. 24 schematically illustrates a process for fixing a tissue samplefor on-CARD processing, according to a non-limiting, illustrative aspectof the invention;

FIG. 25 schematically illustrates an on-CARD sample lysis process, to anon-limiting, illustrative aspect of the invention;

FIGS. 26A-B: Primer Extension Protocol. Amino-terminated capture probescontaining the 3′ interrogating nucleotide are covalently linked to amembrane filter. Following amplification, the amplicon product isdenatured and the complementary strand anneals to the probe. If there isa perfect match between the 3′ nucleotide of the probe and the amplicon(A), DNA synthesis will occur incorporating biotinylated (bio)nucleotides. If there is a mismatch between the 3′ nucleotide of theprobe and the amplicon (B), DNA synthesis will not occur and thus noincorporation of biotin. See Example 2 for further details;

FIG. 27: Representative image derived from the primer extensioncomponent of the Warfarin SNP assay on an embodiment of the CARD.Colored precipitates are detected following hybridization of denaturedamplicons with solid-phase capture probes, followed by incorporation ofbiotinylated dUTP and non-labeled dCTP, dATP, and dGTP in the presenceof DNA polymerase, and finally incubation with streptavidinylated HRPand substrate. The key indicates the allele targeted by the captureprobe. Following imaging, the number of pixels detected on the WT spotis divided by the number of pixels detected on the mutant spot. WTpixels/mut pixels>1 indicates a homozygous WT; WT pixels/mut pixels=1indicates a heterozygous; and WT pixels/mut pixels<1 indicates ahomozygous mutant. The resulting genotype indicated on the filter is:VKORC1—heterozygote; CYP2C9*2—homozygous WT; and CYP2C9*3—homozygous WT;

FIG. 28: Results of seven individual genotypes obtained using theWarfarin SNP assay in conjunction with primer extension on an embodimentof the CARD. Filter results from buccal swabs of seven individuals areshown in the upper portion of the figure. The lower portion of thefigure indicates the genotypes read from these filters. In addition, allgenotypes were verified via bi-directional sequencing. These genotypeswere represented within the 20 individuals tested. The more rarecombinations of genotypes were not expected in this small sample set;

FIG. 29: Results of individual genotypes obtained using the Warfarin SNPassay in conjunction with RDB on an embodiment of the CARD. Filterresults from buccal swabs of four individuals are shown in the upperportion of the figure. The table below the filters indicates thegenotypes read from the filters. The filter key at the bottom of thefigure shows the spotting system within each of the four microarrays.

FIG. 30, Table 1: Capture probe sequences, SEQ ID NOS:1-20. See Example4 for details;

FIG. 31, Table 2: Forward and reverse primers with type specific L1sequences, SEQ ID NOS:23-46. See Example 4 for details;

FIG. 32, Table 3A: Forward primers for amplifying the HPV L1 gene, SEQID NOS:50-77. See Example 4 for details;

FIG. 33, Table 3B: Biotinylated reverse primers for amplifying the HPVL1 gene, SEQ ID NOS:78-105. See Example 4 for details;

FIG. 34: Validation of HPV amplification with L1 primer mix. Full lengthHPV containing plasmids, types 6, 11, 16, 18, and 52, and partial HPVtype 35 containing plasmid were subjected to amplification with the L1primer mix. PCR was performed for 40 cycles on 1000 copies of plasmidDNA on a background of 16 ng C33A nucleic acid. M=100 bp DNA. SeeExample 4 for details;

FIGS. 35A-D: PCR amplification of serially diluted HPV clones. A)Representative images of gels used to separate products resulting fromamplification of 2 fold increasing amounts of full-length HPV 16 andHPV-18 containing plasmids; Lanes are as follows, Marker (100 bp DNALadder), 0, 122, 244, 488, 977, 1,953, 3,906, 7,813, 15,625, 31,250,62,500, 125,000 copies of HPV plasmids. Lower image in A is arepresentative gel showing the separation of products resulting from theamplification of globin in the equivalent samples shown in the upperimages. Specifically, this is the companion gel to the HPV 16 imageshown above. B) Image analysis of gels separating PCR products offull-length (6, 11, 16, 18, 52) or partial (35) HPV containing plasmids.C) Image analysis of gels separating PCR products of HPV L1 clonesgenerated from clinical samples. D) Image analysis of the companionglobin gel for each of the curves shown in A. The bars represent theaverage and standard deviation of globin amplification in the presenceof the different number of input HPV molecules. See Example 4 fordetails;

FIG. 36: PCR amplification of beta-globin in serially diluted C33Apurified nucleic acid. Upper panel: Representative image of gels used toseparate products resulting from amplification of beta-globin in 2 foldincreasing amounts of C33A purified nucleic acid. M=Marker (100 bp DNALadder), 0, 0.025, 0.5, 1, 2, 4, 8, and 16 correspond to total inputnucleic acid in ng. Lower panel: Image analysis of gels separatingproducts following amplification for 30 (N=4), 35 (N=3) or 40 (N=2)cycles of PCR. Data points are average+/−standard deviation. See Example4 for details;

FIGS. 37A-B: A. Results of amplification of 10 copies/μl of HPV over abackground of 1.6 ng/μl genomic nucleic acid. The upper image shows thegel image of HPV amplicons, and the bottom image shows the correspondingglobin amplicons. B. Results of RDB of each combination of HPV andglobin amplicon. See Example 4 for details;

FIG. 38: Nucleic acids purified from clinical samples. Red bars, ng/ulDNA yields based on absorbance at 260 nm (left Y-axis). Blue curve:pixel intensity obtained from image analysis of gels separating productsof PCR amplification of beta-globin (right Y-axis). Amplification wascarried out on 1 μl of purified nucleic acid, regardless ofconcentration, for 30 cycles. Linear trendlines for both data sets arealso shown. See Example 4 for details:

FIGS. 39A-C: Estimation of genomic equivalents in clinical samples.Amplification of beta-globin was carried out on diluted purified nucleicacid from selected clinical samples. A) Standard curve was generatedusing RNA free C33A DNA RNA-DNA. B) and C) Image analysis was used tocalculate the equivalent “genome” based on the line equation shown inthe inset in A (rear bars). Actual input nucleic acid based onabsorbance at 260 nm (front bars) is also shown. See Example 4 fordetails;

FIG. 40, Table 4: Summary of results of PCR and RDB of the clinicalsamples. As shown in the table, 75% (88/117) of the samples demonstratedan HPV amplified band following PCR and 45% (40/88) of the HPV ampliconpositives were captured by an HPV-specific probe on RDB. See Example 4for details;

FIGS. 41A-B: A: Comparison of samples positive for HPV high risk usingthe Digene system versus the HPV result obtained with the embodimentdescribed in Example 4; B: 6 high risk samples were detected by theembodiment described in Example 4 that were read as negative by theDigene system, demonstrating the superiority of the Example 4 embodimentwith respect to sensitivity and specificity of the assay. See Example 4for details;

FIG. 42: The sequence of the L1 gene of HPV 16 (SEQ ID NO:106)containing highlighted sequences corresponding to the various targetregions for amplification (SEQ ID NOS:107-112). See Example 4 fordetails;

FIGS. 43A-C: A: Results of plotting increasing HPV pixel intensity withthe corresponding globin pixel intensity; B: The ratio of HPV to globinpixels plotted versus low to high risk HPV types; C: The ratio of HPV toglobin pixels plotted versus low to high risk HPV types. The “Digene”series plots the numbers which are the results of the commerciallyavailable Digene assay in comparison to with the present assay. Theinset chart shows the Digene assay results (with a different scaleY-axis) that were then scaled and incorporated in the main graph. SeeExample 4 for details;

FIG. 44 shows results obtained from the CARD-based detection of a sparsetarget with lateral flow assay described in Example 5. Heat shock mRNAfrom Cryptosporidium parvum was detected. IMS=immunomagnetic separation.HS=heat shock;

FIG. 45 shows that using the CARD configuration shown in FIG. 20, andaccording to the process shown in FIGS. 21-22, DNA was successfullypurified, eluted and amplified;

FIG. 46 shows an alternative perspective view of a self-containedbiological assay apparatus including an optical system for theperformance of real-time PCR analysis of nucleic acid amplificationreactions (for clarity certain elements of the dispensing system havebeen removed), according to an exemplary embodiment of the invention;

FIG. 47 shows a schematic view of an optical system suited to generate awide spectrum of source light and various dichroic mirrors suited toseparate the fluorescent signal emitted by the fluorophores in thereal-time PCR reaction into particular wavelengths for analysis,according to an exemplary embodiment of the invention;

FIG. 48a and FIG. 48b show a cross sectional view and a schematic viewof an alternative component of an optical system. The alternative systemincludes a single light source with a single detection photodiode. Usingthe alternative system requires multiple optical modules installed onthe instrument—one for each fluorophore expected to be used in thereal-time PCR reaction, according to an exemplary embodiment of theinvention;

FIG. 49 shows a layered cross sectional top plan view of a of a “fourassay unit” arrangement (in all respects identical to FIG. 14A with onlythe addition of optical channels), according to an exemplary aspect ofthe invention;

FIG. 50 shows a perspective view of a “four assay unit” component ofFIG. 49 including the reservoir layer, the fluidic transport layerincluding a film layer, optical channels and amplification reactors,according to an exemplary aspect of the invention;

FIG. 51 shows a cross sectional view of the “four assay unit” componentshown in FIG. 49, according to an exemplary aspect of the invention;

FIG. 52a shows an exploded view of an exemplary “four assay unit”component including optical channels, the reservoir layer, the fluidiclayer, the film layer, the amplification reactors including lumens whichare used to fill and empty the amplification reactors, the membrane forthe analysis reservoir, a silica filter and a perforated retaining ring,according to an exemplary aspect of the invention.

FIG. 52B shows an alternative exploded view of an exemplary “four assayunit” component including optical channels, a monolithic fluidic layerwith integrated reservoirs, the film layer, the amplification reactorsincluding lumens which are used to fill and empty the amplificationreactors, the membrane for the analysis reservoir, a silica filter and aperforated retaining ring, according to an exemplary aspect of theinvention.

DETAILED DESCRIPTION

Reference will now be made in detail to the present exemplaryembodiments of the invention, examples of which are described below andfurther illustrated in the accompanying drawing figures. Whereverpossible, the same reference numbers will be used throughout the figuresto refer to the same or like parts.

FIGS. 1-4, illustrate a self-contained, fully automated, biologicalassay apparatus 10 according to an embodiment of the invention. Theapparatus includes a housing 11; a dispensing platform 12 including acontrollable, movable reagent dispensing system 13 disposed in thehousing; a reagent supply component 14 that can hold a supply of one ormore reagents, in operative connection with the dispensing system,disposed in the housing; a pneumatic manifold 15 that operates to affectthe transport of fluid in a fluidic transport layer 16 and a pluralityof reservoirs 17 attached to the fluidic transport layer 16 opposite thepneumatic manifold 15, that is removably disposed in the housing 11 in aspace shared by the dispensing platform 12, wherein the fluidictransport layer 16, reservoirs 17 and a test sample to be introducedtherein are disposed in a space separate from the dispensing platform12; a pneumatic supply system 18, which functions to provide pneumaticsignals (positive and negative (vacuum) pressure) routed through thepneumatic manifold 15 to affect the transport of the test sample and thereagents in the fluidic transport layer 16, and its plurality ofreservoirs 17, disposed on the pneumatic manifold 15 and in operativeconnection with the pneumatic supply system 18 in the housing 11 in aspace separate from the dispensing platform 12; and a control system 19that is used to control the dispensing platform 12, the heating systems(29, 48, and 48 a), the cooling system (accomplished by directing a jetof compressed air, that may or may not be actively cooled, from thepneumatic supply system 18 at the heater, or supplying airflow from afan) and the pneumatic supply system 18, disposed in the housing 11.

With reference to FIGS. 1-4, 5, 6, 9, 10-13, 16 and 20, aself-contained, biological assay apparatus 10 is designed to require anoperator of the assay apparatus 10 only to introduce a biological sampleof interest into a specific sample input port 33 of a plurality of suchports 33 _(n) on the reservoir layer 17, which covers the fluidictransport layer 16 (with attached film layer 30) and, which altogetherare managed by the pneumatic manifold 15, and then initiate a startsequence of the control system 19, whereby the sample is completely andautomatically processed through an assay to the assay's end point. Asdepicted in the figures, there are 24 separate exemplary sample assayunits arranged in a “six by four” pattern, where each sample isseparate, within its assay unit, from all other samples, and from thebiological assay apparatus throughout the processing sequence. Although24 assay units are depicted in the exemplary embodiment, the lower limitis one assay unit and there is no particular upper limit.

In operation, once a sample to be analyzed has been introduced into thesample input reservoir 33 of reservoir layer 17, the sample isautomatically drawn from the sample input reservoir into the fluidictransport layer 16, or reagents are transported by the fluidic transportlayer 16 from a separate reservoir to the sample input reservoir 33. Allof the processing steps required to analyze the sample are then carriedout within either the reservoir layer 17 and/or the fluidic transportlayer 16 including amplification via appropriate reactors (e.g., 31,FIGS. 11, 12) disposed in the fluidic layer 16 or communicating with thefluidic layer 16 via a lumen 44 of the system, without the interventionof an operator. In completing all of the series of processing stepsrequired to analyze the sample automatically, the system maintainsseparation of the sample from the reagent dispensing system 13, thepneumatic manifold 15, and the pneumatic supply system 18. Thisseparation feature eliminates cross-contamination problems thatinevitably increase the costs and decrease the reliability of currentsystems that perform similar tasks but that require the sample move fromstation to station within an apparatus while it is being processed orthat require the dispensing system to impart movement of fluids within amicrofluidic system.

The apparatus 10 includes a housing 11 that is designed to enclose theinterior of the system during operation so that the fully automatedprocessing can proceed to completion uninterrupted by an operator. Thehousing 11 also encloses the control system 19, the dispensing platform12, the reagent supply component 14, and the pneumatic supply system 18,which operates the pneumatic manifold 15.

FIGS. 1-4 also depict the reagent dispensing system 13. The reagentdispensing system is attached to the X-Y-Z motion control system andtogether is the dispensing platform 12. The dispensing system 13 iscomprised of a dispenser needle 23, a large storage loop 25, a smallstorage loop 26, a solenoid valve 24 for each storage loop 25, 26, and acamera 27. The dispenser needle 23 is comprised of a double barrel tubeor elongate needle structures of length, L, having different respective,selected bore sizes. The smaller barrel (bore) is attached to the smallstorage loop 26 through a dedicated solenoid valve 24 and the largerbarrel is attached to the large storage loop 25 through a dedicatedsolenoid valve 24. The purpose of the dissimilar size is to facilitateprecise metering of reagents delivered by the reagent dispensing system13. The smaller the bore of the barrel of the needle, the easier it isto meter smaller amounts of reagent. In an illustrative aspect, one ofthe reagent dispenser needles has a bore diameter b₁, where0.003≦b₁≦0.018 inches (in) and the other reagent dispenser needle has abore diameter b₂, where 0.015≦b₂≦0.030 in.

The camera 27 may have various functions for the self-containedbiological analysis system 10. One function may be to coordinate thelocation of the dispenser needle 23 of the dispensing system 13 with thereagent supply component 14 and the reservoir layer 17 attached to thefluidic transport layer 16 so the reagents in the reagent supplycomponent 14 are dispensed into the proper reservoir on the reservoirlayer 17. Another function is to provide sample and/or analysisinformation to the control system 19. When an operator provides a sampleto the sample input port 33 of reservoir layer 17 of a particular assayunit the camera 27 can record the sample's information from an opticalsource such as a bar code or other distinct optical marking systemsknown in the art. The information from the sample can then inform thecontrol system 19 of proper sample loading and later it may be combinedwith the resulting analysis. The identified and properly loaded samplethen is processed by the self-contained biological assay apparatus 10and the end result recorded by the same camera 27. The information canthen be communicated to the operator through a control system operatorinterface.

The small and large control loops 25, 26 are coils of tubing attachedthrough the solenoid valves 24 to their particular barrel of the doublebarrel dispensing needle 23. When a particular reagent is required by anassay the dispensing needle 23 is moved by the X-Y-Z motion controlsystem to the particular reagent in the reagent supply component 14. Theneedle is inserted into the reagent's container and negative pressure issupplied through the small or large storage loop 25, 26 and through anopen solenoid valve 24. The desired amount of the particular reagent iswithdrawn from the reagent's container. The X-Y-Z motion control systemthen transports the dispensing system 13 to the location of a reservoirrequiring the reagent. Positive pressure is then supplied to the largeor small storage loop and the appropriate solenoid valve is opened forit to dispense a metered amount of reagent into the reservoir. Thedispensing system 13 may then be repositioned to another reservoirrequiring the same reagent and the dispensing process repeated until allof the reservoirs requiring a particular reagent are supplied. Thedispensing needle 23 and the storage loop 25 or 26 used are then cleanedby repeated flushing of the dispensing needle 23 and the tubing of thestorage loop 25 or 26 with the appropriate washing fluid. The dispensingsystem 13 is then prepared to transport and supply another reagent whenthe assay requires it.

The proper metering of reagents is accomplished by active controllingthrough control system 19 of the positive or negative pressure suppliedto the storage loops 25, 26 and timing the opening and closing of thesolenoid valves 24 of the dispensing system 13 while either withdrawingor dispensing reagents.

The deployment of a single dispensing needle 23 that is automaticallycleaned is an advantage since alternative methods of reagent dispensinggenerally require the use of a pipetter and large numbers of disposablepipette tips. The self-contained biological assay apparatus 10 uses afluidic transport layer 16 that separates the sample from the reagentinput reservoirs in the reservoir layer 17 thus avoiding the potentialfor cross contamination that occurs in alternative systems. Thereforethe single dispensing needle system 23 is employed and the need for apipetter system is not required. Alternatively, the dispenser system maybe configured as a pipetter system and operate in a similar manneralthough incorporating disposable pipette tips instead of cleaning thedispenser needle between reagent applications. In the case where apipette system is employed the sample may also be automaticallydispensed into the sample input port 33 by the pipette and the pipettetip disposed to avoid cross contamination.

As variously illustrated in FIGS. 5-14, the pneumatic manifold 15, whichis comprised of two subunits 15 a and 15 b where subunit 15 a receivesthe pneumatic supply from the pneumatic supply system 18 and splits thesignals through its integrated pneumatic channels 32 to deliver thepneumatic signals to subunit 15 b's pneumatic channels that in turnsupply the pneumatic signals to the diaphragms on the underside of thefluidic transport layer, interfaces with a plurality of microfluidicsystems (16, 16 a, and 17 in combination, referred to hereinafter as anassay unit or, commercially, as a CARD® (Chemistry and Reagent Device)),each CARD having a singular or plural assay capacity. Each CARD furtherincludes a multi-layer, monolithic, polymeric, non-elastomericmicrofluidic chip (microfluidic transport layer) 16 that has a givenconfiguration of microfeatures including a plurality of pneumaticsignal-actuated diaphragm valves. The system further includes aseparate, replaceable pneumatic manifold including a plurality ofpneumatic ports there through and a plurality of pneumatic channelsdisposed therein in fluid connection with both the plurality ofdiaphragm valves and the plurality of pneumatic ports. The plurality ofpneumatic ports and pneumatic channels have a given configurationspecifically corresponding to the given configuration of the pluralityof diaphragm valves on the fluidic transport layer 16. The fluidictransport layer 16 is removably connected to the pneumatic manifold 15.The pneumatic supply system 18 further comprises a plurality ofpneumatic connections that provide pneumatic signals to the pneumaticmanifold 15, in fluid connection with the plurality of pneumatic ports.The plurality of pneumatic connections have a configurationcorresponding to the configuration of the plurality of pneumatic portsof the pneumatic manifold 15. The pneumatic manifold 15 is removablyconnected to the pneumatic supply system 18.

Each multi-layer, monolithic fluidic transport layer 16 further includesa polymeric, non-elastomeric substrate 16 a having a plurality of fluidchannels 39 disposed therein, each of the fluid channels 39 having aninlet end and an outlet end, and at least one bi-directional diaphragmpump comprising at least three non-elastomeric membrane-based valvestructures that are constructed from a single, non-elastomeric,polymeric film layer 30. In various aspects, each fluidic transportlayer 16 may include an integral or component reservoir layer 17including at least a sample input reservoir 33 capable of holding asample and at least reagent reservoir (34) that is capable of holding areagent material.

The reservoir layer 17 and its attached fluidic transport layer 16(including transport layer substrate 16 a) are removably disposed on thepneumatic manifold 15 so that upon completion of an analysis thecombined reservoir layer 17 and fluidic transport layer 16 may beremoved and replaced with a different combined reservoir layer 17 andfluidic transport layer 16 that is either unused or has been cleaned andprepared for re-use. The pneumatic manifold 15 is also removablydisposed on the pneumatic supply system 18 in the housing. The pneumaticmanifold 15 may also then be replaced with another pneumatic manifold 15that is complimentary to another arrangement of combined reservoir layer17 and fluidic transport layer 16 designed for alternative assays orgreater or lesser numbers of assay units of any particular assay.

As illustrated in FIGS. 7, 14C, 15A, 15B, 18 and 19, the pneumaticmanifold 15 may include multi-purpose and/or specific purpose heaters(29, 48 and 48 a) used, e.g., to heat the amplification/reactionreactors 31 and/or other reservoirs or channels. In a non-limitingaspect as illustrated in an exploded view in FIG. 23, the multipurposeheater 2301 may be in the form of a laminated copper/aluminum structure2303 that is mounted on spring-loaded electrical contact pins 2305,which provide electrical contact for resistors 2307 that generate heatthat is then conducted through the laminated heater body. The contactpins also provide a connection to a temperature sensing device 2309 usedto modulate the heater. The spring-mounted electrical contacts alsoprovide uniform contact between the heater surfaces and the object to beheated. The laminated heater apparatus may be a commercially availablemetal core (laminated copper/aluminum; e.g., Thermally Conductive PCBSubstrate, Laird Technologies, Chesterfield, Mo.) board. The metal coreboard provides excellent thermal conductivity, which further providesgood temperature uniformity with a temperature sensor component builtinto the circuit layout. One of the challenges of microfluidic systemsis being able to accurately measure the temperature of the heated area.The pogo contact pins (e.g., Spring Contact Probes, Interconnect DevicesIncorporated, Kansas City, Kans.) provide electrical and mechanicalinterface. The fluidic transport layer 16 interfaces with the pneumaticports and the heaters 29, 48 and 48 a. When the diaphragm pumps disposedon the fluidic layer 16 are operated by positive and negative pneumaticpressure supplied to the pneumatic manifold 15 from the pneumatic supplysystem 18, the sample and/or reagents are drawn into the fluidictransport layer 16 and transported there through to undergo variousprocessing steps and analysis steps, involving the reservoir layer 17,amplification reactor(s) 31, and other components such as the heaters29, 48 and 48 a and magnets 49 disposed on the pneumatic manifold 15.Throughout the process the sample and any intermediary reactionsperformed in the fluidic transport layer 16, the reservoir layer 17, oran amplification reactor 31 are separated in their particular assay unitfrom the other assay units. The separation prevents cross contaminationof the sample with or from any other assay units, the reagent dispensingsystem 13, the pneumatic manifold 15, or the pneumatic supply system 18,thereby providing greater analytical reliability and decreased operationcosts since the system's cleaning requirements are greatly reduced. Thepneumatic supply system 18 functions to provide pneumatic signals(positive and negative pressure) routed through the pneumatic manifold15 to affect the transport of the test sample and the reagents withinthe fluidic transport layer 16. Its operation is coordinated with thedispensing platform 12 so that when reagents are delivered to particularor common reservoirs on reservoir layer 17 by the dispensing system 13from the reagent supply component 14 the proper processing proceduresoccur. The pneumatic manifold 15 also may incorporate areas whereheating, cooling (accomplished by directing a jet of compressed air,that may or may not be actively cooled, from the pneumatic supply system18 at the heater, or supplying air flow from a fan), or magnetism may beapplied to facilitate a reaction. The pneumatic supply system 18 isdisposed in operative connection with the pneumatic manifold 15 in thehousing 11 in a space separate from the dispensing platform 12 and acontrol system 19 that is used to control the dispensing platform 12,the pneumatic supply system 18, and any heating, cooling (accomplishedby directing a jet of compressed air, that may or may not be activelycooled, from the pneumatic supply system 18 at the heater or supplyingair flow from a fan), or magnetism performed by the pneumatic manifold15 reaction occurring in the fluidic transport layer 16, the reservoirlayer 17 or the amplification reactors 31.

FIG. 4 is a perspective view of the self-contained biological assayapparatus 10, which more clearly shows the location of the pneumaticsupply system 18 beneath the pneumatic manifold 15. The pneumatic supplysystem 18 is located within the lower portion of the housing 11. Thepneumatic manifold 15 is removably attached to the pneumatic supplysystem 18 in order to facilitate easy reconfiguration of theself-contained biological assay unit 10 for assays that requiredifferent designs of the fluidic transport layer 16. The pneumaticsupply system 18 is comprised of a number of subcomponents designed tostore, meter and route pneumatic pressure under the direction of thecontrol system 19 through the pneumatic manifold 15 to operate thediaphragms located on the fluidic transport layer 16 of an assay unit.Positive and negative pressure is supplied to the positive and negativepressure storage reservoirs 21 through positive and negative pressureregulators 20. Positive and negative pressure are in turn metered andsupplied to the channels of the pneumatic manifold 15 from the positiveand negative pressure storage reservoirs 21 through pneumatic supplysolenoids 22 (the tubes leading from the pneumatic supply solenoids 22to the undersurface of the pneumatic manifold 15 are not shown forclarity (the configuration may also be configured without tubes byhaving a tubeless solid state interface between the pneumatic supplysystem 18 and the pneumatic manifold 15)). The pressure metering and theopening and closing of the solenoids is managed by control system 19 andsupplies the metered pneumatic force to the pneumatic manifold 15,thereby operating the diaphragm pumps located on the fluidic transportlayer 16.

FIG. 5 illustrates an array of six CARDs depicting a “six by four”arrangement of reservoirs, each four assay unit CARD being removablydisposed on the pneumatic manifold 15. In operation, the reservoir layer17 is covered by a film (not shown) to protect its contents frompotential cross contamination by samples in process in adjacent assayunits and to prevent the contents of the reservoirs 17 fromcontaminating the dispensing platform 12, the pneumatic manifold 15, thepneumatic supply system 18, the control system 19, or the housing 11.

FIG. 6 further illustrates a “six by four” CARD arrangement removablyattached to the fluidic transport layer 16 on the pneumatic manifold 15.

FIG. 7 is a top view of the interface between the fluidic transportlayer 16 and the pneumatic manifold 15. Gaskets 28 are attached to thetop of the pneumatic manifold 15 and are designed to isolate thepneumatic forces applied to the film layer 30 (FIG. 15B) of the fluidictransport layer 16. Terminating within each gasket void is a channelrouted through the pneumatic manifold 15 from the pneumatic supplysystem 18. When a fluidic transport layer 16 is placed on top of thegasket 28, the unbonded portion of the film layer 30 of the fluidictransport layer 16 is then able to be flexed into or out of the gasketvoid by applied positive and/or negative pressure routed through thepneumatic manifold 15 from the pneumatic supply system 18 and controlledby control system 19. The control system 19 can apply the positive ornegative pressure sequentially to selected locations, which creates adiaphragm pumping system as described in commonly assigned U.S. Pat. No.7,832,429. By using such a system of actuated pumping, the pneumaticmanifold supply system 18 and the pneumatic manifold 15 remain separatedfrom the fluids transported through the fluidic transport layer 16,which eliminates cross-contamination between assay units andcontamination of the pneumatic supply system 18 and the pneumaticmanifold 15.

As mentioned above, heaters 29 may be located within the pneumaticmanifold 15 that the amplification reactors 31 fit into when the CARD isplaced onto the pneumatic manifold 15. At the appropriate time duringthe processing of a sample, the amplification reactor 31 is filled withthe appropriate reagents and processed sample transported through thefluidic transport layer 16 from various reservoirs in reservoir layer17. The contents of the amplification reactor are then heated and cooled(accomplished by directing a jet of compressed air, that may or may notbe actively cooled, from the pneumatic supply system 18 at the heater orsupplying air flow from a fan) in a controlled manner throughinstructions from the control system 19.

FIG. 8A illustrates a perspective view of the layer 15 a of a “six byfour” arrangement of the pneumatic manifold 15 of the self-containedbiological assay apparatus 10. The orifices through which the pneumaticsignals are routed to higher layers 15 b of the pneumatic manifold 15and the bays where the heaters are located.

FIG. 8B illustrates a top layered view of a “six by four” arrangement ofthe pneumatic manifold 15 a of the self-contained biological assayapparatus 10. The channels 32 through which the pneumatic signals arerouted from the orifices on the bottom of the pneumatic manifold 15 formthe pneumatic supply system 18 and the orifices on the layer depicted in8A.

FIG. 8C illustrates a perspective bottom view of a “six by four”arrangement of the pneumatic manifold 15 of the self-containedbiological assay apparatus 10. The pneumatic solenoids 22 and thepressure storage reservoirs 21 depicted in FIG. 4 supply pneumaticpressure or vacuum to the orifices located on the bottom of thepneumatic manifold 15. The pressure storage reservoirs 21 supply meteredpositive and negative pressure to the pneumatic channels 32 shown inFIG. 8B that are fabricated in the pneumatic manifold 15. When thecontrol system 19 opens a pneumatic supply solenoid 22, a pneumaticchannel 32 is either supplied with metered negative or metered positivepressure depending upon which pressure storage reservoir 21 is attachedto the specific pneumatic supply solenoid 22 opened or closed by thecontrol system 19. The metering of the pressure is accomplished byadjusting the pressure of pressure storage reservoirs 21, modulating theopening of pneumatic supply solenoids 22 or a combination of both underthe management of control system 19 The pressure supplied to the channelthen operates the fluidic transport layer 16 through the gasketinterface 28 of the pneumatic manifold 15.

FIG. 9 illustrates a layered top view of a “four assay unit” CARD, withfluidic channels 39 of the fluidic transport layer 16 and amplificationreactors 31. The figure shows sample input reservoir 33, commonpreparative and purification reagent reservoir 34, waste reservoir 35,and silica filter reservoir 36 including silica filter retaining ring 36a, which would hold silica filter 36 b (not shown for clarity) in thepreparation and purification area of a single assay portion of a CARD.It also shows elution reservoirs 37 (left and right), amplificationmaster mix reservoirs 38 (left and right), and amplification reactors 31(left and right) attached underneath the fluidic transport layer 16 andaccessed through a lumen 44. It also shows common reagent inputreservoir 40, analysis reservoir 41, and waste reservoir 42 and theperforated ring system 43 used to support the analysis membrane 43 (notshown for clarity) of the analysis portion of a single assay unit of afour assay unit CARD.

FIGS. 10A through 10K generally illustrate a non-limiting, exemplarymethod of using the device to process and analyze a sample. Adescription of a single assay unit is provided here though there is noparticular upper limit to the number of assay units that can processsamples either in parallel or serially as samples are provided to theself-contained biological assay apparatus 10. The order that individualsamples are processed are based upon the capabilities of the controlsystem 19 to manage the pneumatic supply system 18 and the particulararrangement of the pneumatic manifold 15, and the specific reagentssupplied by the reagent supply component 14 and delivered by thedispensing system 13. In particular, a specific assay may require adesign of a reservoir layer 17 and its matching fluidic transport layer16 that interfaces with the pneumatic manifold 15. When all of thematching elements are combined, the process will generally proceed asfollows.

In FIG. 10A, a sample is input into sample input reservoir 33 and thedispensing system 13 provides a cell lysing reagent into commonreservoir 34. The cell lysing reagent is then pumped to the sample inputreservoir 33 and incubated either with or without gentle agitation asdescribed in co-pending application Ser. No. 12/249,872 as “fluffing”(i.e., the repeated actuation of a diaphragm accessing a reservoir toalternatively withdraw and then inject fluid into a reservoir to causeturbulence and mixing), as required by the assay. While the sample isincubating in sample input reservoir 33, an organic alcohol (e.g.,ethanol) is dispensed by the dispensing system 13 into common reservoir34 and pumped to sample input reservoir 33 further increasing the volumein sample input reservoir 33; the larger mixture continues to incubate.Upon completion of the incubation period the contents of sample inputreservoir 33 are pumped onto the top of the silica filter 36 b in silicafilter reservoir 36 and pulled through the silica filter 36 b and thecontents pumped to waste reservoir 35.

Per FIG. 10B, an organic alcohol (e.g., ethanol) is dispensed intocommon reservoir 34 and pumped on top of the silica filter 36 b insilica filter reservoir 36, pulled through the silica filter and pumpedto waste reservoir 35. A wash buffer is dispensed into common reservoir34 and pumped on top of the silica filter 36 b in silica filterreservoir 36, pulled through the silica filter 36 b and pumped to wastereservoir 35. The same or another wash buffer is dispensed into commonreservoir 34 and pumped on top of the silica filter 36 b in silicafilter reservoir 36, pulled through the silica filter 36 b and pumped towaste reservoir 35.

In FIG. 10C, an elution buffer is then dispensed into common reservoir34 and pumped directly to waste reservoir 35 to clear the channel ofresidual lysate, the organic alcohol, and wash buffer. Fresh elutionbuffer is dispensed into common reservoir 34 and pumped on top of thesilica filter 36 b in silica filter reservoir 36 to clean the reservoir,then it is pumped from the top of the silica filter 36 b to wastereservoir 35.

In FIG. 10D, elution buffer is then dispensed into elution reservoir 37(right) and pumped up through the bottom of the silica filter 36 b inreservoir 36.

In FIG. 10E, amplification master mix is then dispensed intoamplification master mix reservoirs 38 (left and right). An initialvolume from silica filter reservoir 36 is pumped into elution reservoir37 (right), then a single pump of material is pumped from silica filterreservoir 36 to amplification master mix reservoir 38 (right). Thecontents of amplification master mix reservoir 38 (right) are thenemptied into amplification reactor 31 (right). The same process isrepeated on the left side. Then the contents of amplification reactors31 (left and right) are thermocycled under the control of control system19 in accordance with the protocols of the particular assay in process.

Next, per FIG. 10F following thermocycling, a pre-hybridization bufferis dispensed into common reagent reservoir 40 and pumped intoamplification reactor 31 (right), then repeated to providepre-hybridization buffer to amplification reactor 31 (left). Thetemperature in the amplification reactors is then increased to denaturethe amplicons produced from the earlier thermocycling.

In FIG. 10G, in order to prepare the microarray on analysis membrane 47to properly hybridize with the amplicons produced from the earlierthermocycling, while the amplicons are denaturing, dispense the same oranother pre-hybridization buffer into common reagent reservoir 40 andpump it on top of the analysis membrane 47 (not shown for clarity)suspended in analysis reservoir 41, then pump it to waste reservoir 42;dispense wash buffer into common reagent reservoir 40, pump it over theanalysis membrane 47 in analysis reservoir 41, and pump to wastereservoir 42 (repeat multiple times).

In FIG. 10H, the same or another pre-hybridization buffer is dispensedinto common reagent reservoir 40 and from there into amplificationreactors 31 (left and right) and on top of the analysis membrane 47 inanalysis reservoir 41.

In FIG. 10I, the contents of amplification reactors 31 (left and right)are pumped on top of the analysis membrane 47 in analysis reservoir 41and circulated multiple times, which provides sufficient contact betweenthe amplicons and the targets attached to the analysis membrane 47. Whensatisfactory hybridization has occurred, the contents are pumped towaste reservoir 42. Wash buffer is dispensed into common reagentreservoir 40 and pumped on top of the analysis membrane 47 suspended inanalysis reservoir 41, then pumped to waste reservoir 42 (repeatmultiple times).

Next per FIG. 10J, dispense an appropriate amplicon visualizationreagent (e.g., horseradish peroxidase (“HRP”)) into common reagentreservoir 40 and pump on top of analysis membrane 47 suspended inanalysis reservoir 41 and circulate until the reaction is satisfactorilycomplete; then pump the contents to waste reservoir 42. Dispense washbuffer into common reagent reservoir 40 and pump on top of the analysismembrane 47 suspended in analysis reservoir 41, and pump to wastereservoir 42 (repeat multiple times).

Per FIG. 10K, dispense a visualization reagent reactant (e.g.,tetramethyl benzidine “TMB”) into common reagent reservoir 40 and pumpon top of the analysis membrane 47 suspended in analysis reservoir 41;circulate as described above to completely react the reagents (e.g., TMBwith the HRP), and pump the contents to waste reservoir 42. Dispensewash buffer into common reagent reservoir 40 and pump on top of theanalysis membrane 47 suspended in analysis reservoir 41, and pump towaste reservoir 42 (repeat multiple times). Finally, position the camera27 on dispensing system 13 over the analysis membrane 47 suspended inanalysis reservoir 41 and record the image. The image data is thenprocessed by control system 19 and the results communicated to theoperator.

FIG. 11 illustrates a perspective view of a “four assay unit”arrangement of a CARD including the reservoir layer 17, the fluidictransport layer 16 including the film (non-elastomeric membrane ordiaphragm) layer 30, and one of the amplification reactors 31 with itsattachment system 45, 46 as well as a portion of a perforated securingring 43 used to suspend the analysis membrane 47 in analysis reservoir41. More particularly, each exemplary ‘quad’ CARD has four microarrayanalysis reservoirs 41 (FIGS. 9, 11), which include a microarrayanalysis membrane 47 removeably disposed therein. An exemplary analysismembrane 47 is made of nylon, nitrocellulose, PVDF or any otherappropriate material known in the art. The analysis reservoir 41 has asupport platform on the bottom of the reservoir or a shelf or shoulderextending about the bottom inner perimeter thereof. A perforated,segmented, or indented securing ring may be used to secure the membranein the analysis chamber. FIG. 13B shows an exemplary perforated securingring 43 having channels 1708 for transporting fluid from within theanalysis reservoir over the perimeter of the membrane to the bottom ofthe reservoir and then to outside of the reservoir. In an aspect, twosecuring rings 43 may be used to sandwich the membrane 47 therebetweenor as illustrated a support platform slightly smaller than the membranebut not smaller than the inner dimension of the perforated ring 43 maybe used to hold the membrane.

FIG. 12 illustrates a side view of an “assay unit” including thereservoir layer 17, the fluidic transport layer 16 including the filmlayer 30 bounding the channels 39, and one of the amplification reactors31, which includes a lumen 44 used to fill and empty the amplificationreactor.

During a PCR reaction, for e.g., an aqueous solution may be repeatedly(20-50 times) cycled from low temperatures of approximately 30° C. tohigher temperatures of approximately 95° C. In order to prevent theaqueous solution from losing volume due to evaporation or condensation,for e.g., on the tube's side above the bulk solution, it is advantageousto seal the exposed surface of the solution so that the reaction doesnot fail due to lack of sufficient solution volume or changes inreaction concentration from an unsealed environment.

To prevent the evaporation or uncontrolled condensation, wax, silicone,mineral oil, or some other substance may typically be introduced overthe top of the solution to prevent evaporation; however, the use ofthese materials has certain disadvantages. For example, mineral oil is aliquid at room temperature and, therefore, for certain automatic systemsit produces handling problems. Wax is a solid at room temperature andfor automatic systems its melting temperature is very controllable, butwax often impedes the more desirable complex PCR reactions. Silicone,like mineral oil, is a liquid at room temperature, so it has similarhandling problems but it does not impede PCR reactions.

For automatic PCR systems, it would be particularly advantageous to havea substance that could cover the solution automatically when the PCRreaction tube (e.g., 31, FIG. 11, 12) is filled. For example, acontrolled mixture of high purity silicone oil with a small amount (afew %) of wax as an additive may be used. In an exemplary aspect, themixture consists of wax=1% to 20% and silicone oil=99%-80%. According toan aspect, the mixture consists of approximately 5% wax and 95% siliconeoil. The wax may be standard PCR wax (e.g., Sigma Aldrich paraffin waxwith melting point of 58-62 degrees C.). The mixture is a solid at roomtemperature and expected storage temperatures. The mixture may be placedas a layer of material on the upper inside surface of the PCR tube (31)just below the opening of the tube and above the bottom opening of thelumen 44. When the temperature increases during the first thermolcycleis performed after the analyte is introduced to the tube, the mixturemelts and covers the surface of the solution preventing evaporationwhile at the same time it does not have an impeding effect on thereaction. Upon completion of the reaction the reactants can be removedfrom underneath the seal via the lumen (44, FIG. 12) either before orafter the mixture re-hardens upon reducing the temperature in theamplification reactor below the melting point of the mixture. In anaspect, the mixture is allowed to cool forming a solid cap over thesolution and the lumen 44 withdraws the solution from below thesolidified layer.

FIG. 13A illustrates an exploded view of a “four assay unit” CARDincluding the reservoir layer 17, the fluidic transport layer 16including its film layer 30, and the amplification reactors 31, whichinclude lumens 44 used to fill and empty the amplification reactors. Theillustration also shows silica filter holders 36 a, the silica filters36 b for the silica filter reservoirs 36 and the perforated securingrings 43 used to suspend the analysis membranes 47 in the analysischambers.

FIG. 14A-D illustrate an alternative arrangement of a portion of theanalysis area of reservoir layer 17 and the fluidic transport layer 16to which it is attached. The alternative arrangement is used to coverthe analysis membrane 47 in order to improve contact between thecirculating amplicons and the target molecules on the analysis membrane47 in the analysis reservoir 41. The cover may be vented to allow airbubbles to escape from analysis reservoir 41. The arrangement alsoallows for efficient heating of the analysis reservoir 41 by eliminatingthe step or platform used in the perforated ring arrangement 43previously illustrated. In the alternative arrangement the analysismembrane sits directly on the film layer 30 which in turn is disposeddirectly over a heating element 48 a. Heat applied during an analysisreaction is often an important step in an assay. The covered system alsoallows for a different arrangement of the channels supplying andemptying the analysis reservoir. In the alternative arrangement sincethe membrane is on the very bottom of the reservoir the fluid can bewashed over the top of the membrane 47 in an alternating pattern. Inorder to keep the membrane settled on the film layer that constitutesthe bottom of the reservoir an overhang is fabricated in substrate layer16 a of analysis reservoir 41 and as shown in FIG. 14B channel openingsare fabricated under the overhang at each end of the analysis reservoir41. The analysis reservoir 41 is also fabricated so that it is longerthan it is wide which combined with the channel openings onto each endallows for an efficient flow over the length of the analysis membraneand further combined with the cover allows for improved contact of thefluid with the surface of the analysis membrane allowing the ampliconsin the fluid to more efficiently hybridize with their targets attachedto the analysis membrane. The arrangement is further improved byallowing for alternating directions that the fluid can pass over themembrane by circulating the fluid alternatively clockwise and thencounterclockwise using the pumps and channels of the fluidic transportlayer 16. FIG. 14C illustrates an exemplary four assay unit alternativepneumatic manifold 15 and gasket layer 28 that corresponds to thealternative arrangement of the analysis area describe in 14A and 14B.Note the second heater 48 a located on the pneumatic manifold 15underneath the analysis reservoir 41. FIG. 14D illustrates the internalpneumatic channels 32 of pneumatic manifold layer 15 b showing how thepneumatic signals introduced into the bottom layer of 15 a from thepneumatic supply system 18 are further split and addressed to particulargasket layer 28 voids on the surface of 15 b.

FIG. 15A illustrates an exploded perspective view of an exemplary singleassay unit of a pneumatic manifold 15, incorporating pneumaticallyactivated magnet assemblies 51 and a multi-purpose heater 48 coupledthereto, for preparative, amplification, and analysis reaction needs.The figure also includes an optional diaphragm layer 53 for theinterface between the fluidic transport layer 16 and the pneumaticmanifold 15. The illustrated system can be “numbered up” (e.g., “QuadCARD®”) and attached to a pneumatic supply system 18 to operate multipleassay units in parallel or in random order as instructed by the controlsystem 19. The illustrated system provides for the use of magnetism whenrequired during an assay for the manipulation of ferrous, magnetic, orparamagnetic particles as required by any particular assay. In practicethe particle required may be introduced into a particular reservoir onreservoir layer 17 by the dispenser system 13 and combined with thesample during certain steps of the assay. Alternatively, the particlesmay be included with the sample prior to its loading into the sampleinput reservoir 33 or, alternatively still, the particles may bepre-loaded into a reservoir or a channel during the manufacture of thefluidic transport layer 16 or the reservoir layer 17.

The sample with the particles in mixture may be pre-loaded in areservoir or a channel located over a pneumatic piston assembly(alternatively the piston assembly may be motor or electromagneticallyactivated) 51 or the sample/particle mixture may be pumped to areservoir or channel located over a pneumatic piston assembly(alternatively the piston assembly may be motor or electromagneticallyactivated) 51. In either case, the reservoir or channel over thepneumatic piston assembly (alternatively the piston assembly may bemotor or electromagnetically activated) 51 may be subjected to a magnet49, located on a magnet holder 50 fixed on the end of a piston rod 52 ofthe pneumatic piston assembly (alternatively the piston assembly may bemotor or electromagnetically activated) 51 by providing positivepressure to the cylinder of the pneumatic piston assembly (alternativelythe piston assembly may be motor or electromagnetically activated) 51,which then directs the magnet into place just under heated reservoir 55,or non-heated reservoir 56, or a channel containing the sample/particlemixture. When magnetism is delivered to the site with thesample/particle mixture, the assay then takes advantage of theparticles' magnetic properties to carry out particular assayrequirements; e.g., using the particles for a particular concentrationstep of biological material attracted to the particle or other commonparticle dependent assay step known in the art.

Alternatively or in combination, the pneumatically-actuated magnetsystems 51 may be incorporated into a region of the pneumatic manifold15 so that the particles and sample in progress may undergo a heatingevent in conjunction with a magnetism event. An example of such a caseis to use the particles to concentrate an organism out of a largersample volume. In the case where the organism is alive when it iscaptured, it can then be subjected to a heating event to cause theorganism to express RNA that it would not normally express or that itcould not express if it were dead. After the heating event, themagnetism is removed by subjecting the cylinder of the pneumatic pistonassembly (alternatively the piston assembly may be motor orelectromagnetically activated) 51 to negative pressure, thus withdrawingthe magnet 49 from a location underneath heated magnetic reservoir 55,and pumping the solution with the particles and the concentrated samplewith the organism to another location in order to proceed withextracting the RNA from the organism, and further amplifying theextracted RNA and analyzing the resulting amplicons in a mannerconsistent with the process described above.

FIG. 15A also illustrates the opportunity to provide heat in a reservoir54 separate from the amplification reactor 31 in order to denatureamplicons prior to analyzing the amplicons, incubate a labeling reactionor otherwise improve the process of a particular reaction. It furtherillustrates an alternative manner for locating the amplificationreactor. Although this illustration does not provide an amplificationreactor in a tube and lumen 44 arrangement as earlier described, thereis no reason that such a tube and lumen 44 arrangement cannot beutilized with the pneumatic piston assemblies 51 described above. Asshown, the interface between the pneumatic manifold 15 and the fluidictransport layer 16 includes an optional diaphragm layer 53 in place ofthe gasket layer 28 discussed above. The diaphragm layer serves the samefunction of providing an isolated space for the film layer 30 to deflectinto when negative pressure is applied to the unbonded region of thefilm layer 30 by the pneumatic manifold 15 from pneumatic signals routedthrough the pneumatic channels 32 of the pneumatic manifold 15 attachedto the pneumatic supply system 18 under the instructions of controlsystem 19.

FIG. 15B illustrates the same elements as FIG. 15A although without thediaphragm layer. Therefore the gasket layer 28 serves the function asdescribed in FIG. 7 above and the fluidic transport layer 16 interfacesdirectly with the pneumatic manifold 15. The arrangement without thediaphragm layer is advantageous since the manufacturing and the materialcosts in both cases are lower in the absence of the diaphragm layerwhile the performance of assays on the system is no different.

FIG. 16 illustrates a view of a single assay unit for improved rapiddetection of sparse targets, non-viable and viable water-borneorganisms, and shows functional structures including pneumaticallyactivated magnets 51 and multiple heated reaction sites, as well as thearrangement of reservoirs and channels employed by the system. Inparticular, the system illustrates how the pneumatic manifold 15 can bearranged in combination with the fluidic transport layer 16 and itsattached reservoir layer 17 so that heat can be delivered to differentsteps of an assay. In the specific case, heat can be employed in apreparative step in heated magnetic separation/concentration/reactionreservoir 55 (where magnetism can also be selectively employed asdesired in conjunction with heating in reservoir 55). Following optionalheating, the sample is transported through the fluidic transport layer16 for further preparative steps of an assay. Following the extractionof DNA or RNA as required by an assay, the extracted DNA or RNA is thencombined with the amplification master mix and transported through thefluidic transport layer 16 to the amplification reactor 31. Asillustrated in FIG. 16, the reactor is in the plane of the fluidictransport layer 16, though it may also employ a reactor configured as inFIGS. 11, 12 and 13 out of the plane of the fluidic transport layer 16.After the amplicons are produced in the amplification reactor 31, theycan be transported to the heated analysis reactor 54 where they can beheated in order to denature and/or label them, or a combination ofrequirements that may require heating. The amplicons are thentransported to the analysis reservoir 41 (which also can be configuredfor heating) for completion of the assay.

FIGS. 17A-O further illustrate the exemplary analysis processes asfollows:

As illustrated in FIG. 17A, place sample into reservoir 33/55, dispensebuffer and beads into reservoir 33/55 and incubate with gentle agitationby fluffing using the large diaphragm adjacent to reservoir 33/55.Choose temperature and incubation time appropriate for the assay. Raisemagnet 49 into place under reservoir 33/55 and then pump contents towaste reservoir 35.

As illustrated in FIG. 17B, lower magnet 49 out of place under reservoir33/55, dispense a wash buffer into common reservoir 34 (wash buffer 1),and pump wash buffer from common reservoir 34 (wash buffer 1) toreservoir 33/55 to re-suspend the beads. Gently agitate by fluffing asabove, then raise magnet 49 into place under reservoir 33/55 and pumpthe contents to waste reservoir 35.

As illustrated in FIG. 17C, set multi-purpose heater 48 to theappropriate incubation temperature (if required by the particular assay)and, after an appropriate incubation, lower magnet 49 out of place underreservoir 33/55; dispense wash buffer into reservoir 37/56 and pump itto reservoir 33/55 to re-suspend the beads, gently agitate, and setmultipurpose heater 48 to appropriate incubation temperature and time(as required by the particular assay); dispense lysis buffer into commonreagent reservoir 34 (lysis buffer), pump the lysis buffer to reservoir33/55, and agitate by fluffing as above and heat as required by theparticular assay; dispense an organic alcohol (e.g., ethanol) intocommon reagent reservoir 34 (ethanol) and pump it to reservoir 33/55 andagitate by fluffing as above and heat as required by the particularassay.

As illustrated in FIG. 17D, pump the contents of reservoir 33/55 ontothe top of the silica filter 36 b in silica filter reservoir 36.

As illustrated in FIG. 17E, pull the contents of silica filter reservoir36 through the silica filter 36 b in the silica filter reservoir 36 andpump it to waste reservoir 35, followed (optionally) by pulling airthrough the filter by opening vacuum port 59.

As illustrated in FIG. 17F, dispense an organic alcohol (e.g., ethanol)into common reagent reservoir 34 (ethanol) and pump it to silica filterreservoir 36 onto the top of the silica filter 36 b in silica filterreservoir 36; then repeat the steps illustrated in FIG. 17E.

As illustrated in FIG. 17G, dispense the same or another wash bufferinto common reagent reservoir 34 (wash buffer 2) and pump it to silicafilter reservoir 36 onto the top of the silica filter 36 b in silicafilter reservoir 36; then repeat the steps illustrated in FIG. 17E.

As illustrated in FIG. 17H, dispense the same or another wash bufferinto common reagent reservoir 34 (wash buffer 3) and pump it to silicafilter reservoir 36 onto the top of the silica filter 36 b in silicafilter reservoir 36; then repeat the steps illustrated in FIG. 17E.

Repeat the steps illustrated in FIGS. 17H and 17E; then, repeat thesteps illustrated in FIGS. 17F and 17E.

As illustrated in FIG. 17I, dispense wash buffer into reservoir 37/56and pump it to silica filter reservoir 36 onto the top of the silicafilter 36 b in silica filter reservoir 36. Incubate as appropriate andthen pump it back to reservoir 37/56.

As illustrated in FIG. 17J, dispense a binding buffer into reservoir 37(binding buffer) and pump it to reservoir 37/56; then dispense beadsinto reservoir 37/56; incubate and agitate as appropriate. Raise magnet49 into place under reservoir 37/56 and pump the contents of reservoir37/56 to waste reservoir 35.

As illustrated in FIG. 17K, lower magnet 49 out of place under reservoir37/56. Dispense the same or another wash buffer into reservoir 37 (washbuffer A) and pump the contents of reservoir 37 (wash buffer A) intoreservoir 37/56 to re-suspend the beads. Gently agitate and incubate asappropriate. Raise magnet 49 into place under reservoir 37/56 and pumpthe contents of reservoir 37/56 to waste reservoir 35. Repeat multipletimes.

As illustrated in FIG. 17L, lower magnet 49 out of place under reservoir37/56. Dispense the same or another wash buffer into reservoir 37 (washbuffer B) and pump the contents of reservoir 37 (wash buffer B) intoreservoir 37/56 to re-suspend the beads. Gently agitate and incubate asappropriate. Raise magnet 49 into place under reservoir 37/56 and pumpthe contents of reservoir 37/56 to waste reservoir 35. Lower magnet 49out of place under reservoir 37/56. Dispense the same or another washbuffer into reservoir 37 (wash buffer B) and pump the contents ofreservoir 37 (wash buffer B) into reservoir 37/56 to re-suspend thebeads. Gently agitate and incubate as appropriate. Raise magnet 49 intoplace under reservoir 37/56 and pump the contents of reservoir 37/56 toreservoir 37 (wash buffer B) to clear the channel and diaphragms betweenreservoir 37/56 and amplification master mix reservoir 38.

As illustrated in FIG. 17M, lower magnet 49 out of place under reservoir37/56. Dispense amplification master mix into amplification master mixreservoir 38 and pump the contents of amplification master mix reservoir38 into reservoir 37/56 to re-suspend beads. Gently agitate and incubateas appropriate. Pump the contents of reservoir 37/56 back intoamplification master mix reservoir 38. Pump or pull the contents ofamplification master mix reservoir 38 into the amplification reactor andthrough to waste to completely fill the amplification reactor. Incubateat appropriate temperatures and times for amplification.

As illustrated in FIG. 17N, dispense hybridization buffer into analysisreservoir 41 (hybridization buffer) and pump a portion to wastereservoir 42. Pump a portion of the contents of reservoir 41(hybridization buffer) to reservoir 54 (the heated analysis reservoir).Pump a portion of the contents of the amplification reactor to wastereservoir 42. Pump a portion of the contents of the amplificationreactor and another portion of the contents of reservoir 41(hybridization buffer) into reservoir 54 (the heated analysisreservoir).

As illustrated in FIG. 17O, dispense any other required reagents intoreservoir 54 (the heated analysis reservoir) as required. Agitate, heatand incubate the contents of reservoir 54 (the heated analysisreservoir) as required. Pump all or a portion of the contents ofreservoir 54 (the heated analysis reservoir) into reservoir 41 (analysisreservoir) to progress through an analysis of the contents of reservoir54 (the heated analysis reservoir). Dispense running buffer intoreservoir 41 (running buffer) and when all of the contents earlierpumped to reservoir 41 (analysis reservoir) are consumed pump thecontents of reservoir 41 (running buffer) into reservoir 41 (analysisreservoir) to complete the assay. Finally, optically analyze the resultsdisplayed on the filter in reservoir 41 (analysis reservoir).

FIG. 18 illustrates a side cross sectional view of the pneumaticmanifold 15 with integrated pneumatic piston assemblies 51, one of whichis located in the multi-purpose heater assembly 48 and one of which islocated in the preparative area of the system. The figure specificallyshows the internal components of the pneumatic piston assembly(alternatively the piston assembly may be motor or electromagneticallyactivated) 51 for the preparative area of the system.

FIG. 19 illustrates a side cross sectional view of the pneumaticmanifold 15 with integrated pneumatic piston assembly (alternatively thepiston assembly may be motor or electromagnetically activated) 51located in the multipurpose heater assembly 48, and its internalcomponents.

FIG. 20 shows a layered top view of a double assay unit arrangementwhich shows exemplary alternative functions of the pneumatic manifold 15incorporating a pneumatically activated magnet assembly 51 targeting amagnetic separation/concentration channel area 57 instead of a reservoiras described in FIGS. 17A-O, and including the arrangement of thereservoirs and channels employed by the system.

FIGS. 21A-K further illustrate the exemplary analysis processes asfollows:

As illustrated in FIG. 21A, place sample into sample input reservoir 33,dispense enzyme into common reagent reservoir 34 and pump it to sampleinput reservoir 33. Incubate with gentle agitation. Dispense lysisbuffer into common reagent reservoir 34 and pump it to sample inputreservoir 33. Incubate with gentle agitation. Dispense beads into commonreagent reservoir 34 and pump it to sample input reservoir 33. Incubatewith gentle agitation. Dispense an organic alcohol (e.g. isopropanol)into common reagent reservoir 34 and pump it to sample input reservoir33 and incubate it with gentle agitation.

As illustrated in FIG. 21B, raise magnet 49 into place underseparation/concentration channel 57 and pump the contents to wastereservoir 35. Lower magnet 49 out of place underseparation/concentration channel 57. Dispense a wash buffer into commonreagent reservoir 34 (preparation), pump to separation/concentrationchannel 57, and circulate alternatively clockwise and thencounterclockwise multiple times through separation/concentration channel57 to re-suspend and wash the beads. Raise magnet 49 into place underseparation/concentration channel 57 and pump the contents to wastereservoir 35. Lower magnet 49 out of place underseparation/concentration channel 57. Dispense the same or another washbuffer into common reagent reservoir 34 (preparation), pump toseparation/concentration channel 57, and circulate alternativelyclockwise and then counterclockwise multiple times throughseparation/concentration channel 57 to re-suspend and wash the beads.Raise magnet 49 into place under separation/concentration channel 57 andpump the contents to waste reservoir 35. Continue the above wash stepsas required by the assay. Dispense wash buffer into common reagentreservoir 34 (preparation) and pump it to waste reservoir 35 to clearany residual reagents from the reservoir, channels, and diaphragms ofthe preparation area.

As illustrated in FIG. 21C, pump the solution fromseparation/concentration channel 57 to common reagent reservoir 34(preparation). Dispense a high salt buffer into common reagent reservoir34 (preparation). Dispense an organic alcohol (e.g., ethanol) intocommon reagent reservoir 34 (preparation). Pump the contents of commonreagent reservoir 34 (preparation) onto the top of the silica filter 36b in silica filter reservoir 36, pull the contents through silica filter36 b in silica filter reservoir 36, and pump to waste reservoir 35.

As illustrated in FIG. 21D, dispense the same or another wash bufferinto common reagent reservoir 34 (elution), pump onto the top of thesilica filter 36 b in silica filter reservoir 36, pull the contentsthrough silica filter 36 b in silica filter reservoir 36, and pump towaste reservoir 35. Repeat with the same or an alternative wash bufferas required by the assay.

As illustrated in FIG. 21E, dispense elution buffer into reservoir 37(elution) and pump it up through the bottom of the silica filter 36 b insilica filter reservoir 36.

As illustrated in FIG. 21F, pull the contents of silica filter reservoir36 through the silica filter, pump it to elution reservoir 37 (center),and then equally to elution reservoir 37L and 37R; or pump it directlyin equal amounts to reservoir 37L and 37R by bypassing elution reservoir37 (center).

As illustrated in FIG. 21G, dispense amplification master mix intoreservoirs 38L and 38R. The master mix dispensed into the separatereservoirs may or may not contain the same primers depending upon theassay. In the depiction, the original sample is now split into twoaliquots. The sample may remain as a single aliquot or be split intomore than two aliquots depending on the assay in particular and thelayout of available reservoirs, channels, and amplification reactors toaccommodate further splitting of the sample. Pump the solution inreservoir 37L and amplification master mix reservoir 38L into theamplification reactors 31L and 31R (two are depicted for the left sideof the assay unit though only a single amplification reactor isnecessary and more than two are possible). Repeat for the right side ofthe assay unit. Incubate at appropriate temperatures and times foramplification.

As illustrated in FIG. 21H, dispense pre-hybridization buffer intocommon reagent reservoir 40 (left and right) and from there intoamplification reactors 31 (left and right) and on top of the analysismembrane 47 (left and right) in analysis reservoir 41 (left and right).

As illustrated in FIG. 21I, pump the contents of amplification reactors31 (left and right) on top of the analysis membrane 47 (left and right)in analysis reservoir 41 (left and right) and circulate it multipletimes which provides enough contact between the amplicons and thetargets attached to the analysis membrane 47 (left and right). Whensatisfactory hybridization has occurred, pump the contents to wastereservoir 42. Dispense wash buffer into common reagent reservoir 40(left and right) and pump on top of the analysis membrane 47 (left andright) suspended in analysis reservoir 41 (left and right) and pump towaste reservoir 42 (repeat multiple times).

As illustrated in FIG. 21J, dispense an appropriate ampliconvisualization reagent (e.g., horseradish peroxidase (“HRP”)) into commonreagent reservoir 40 (left and right), pump on top of analysis membrane47 (left and right) suspended in analysis reservoir 41 (left and right),and circulate until the reaction is satisfactorily complete, then pumpthe contents to waste reservoir 42. Dispense wash buffer into commonreagent reservoir 40 (left and right), pump on top of the analysismembrane 47 (left and right) suspended in analysis reservoir 41 (leftand right), and then pump the contents to waste reservoir 42. Repeatmultiple times.

As illustrated in FIG. 21K, dispense a visualization reagent reactant(e.g., tetramethyl benzidine “TMB”) into common reagent reservoir 40(left and right), pump on top of the analysis membrane 47 (left andright) suspended in analysis reservoir 41 (left and right), circulate asdescribed above to completely react the reagents (e.g., TMB with theHRP), and pump the contents to waste reservoir 42. Dispense wash bufferinto common reagent reservoir 40 (left and right), pump on top of theanalysis membrane 47 (left and right) suspended in analysis reservoir 41(left and right), and pump to waste reservoir 42 (repeat multipletimes). Finally, position the camera 27 on dispensing system 13 over theanalysis membrane 47 (left and right) suspended in analysis reservoir 41(left and right) and record the image. The image data is then processedby control system 19 and the results communicated to the operator. FIGS.24-25 illustrate a non-limiting, exemplary process for automaticallyanalyzing a tissue specimen using a CARD apparatus as described herein.FIG. 45 shows that using the CARD configuration shown in FIG. 20, andaccording to the process shown in FIGS. 21-22, DNA was successfullypurified, eluted and amplified. The bands shown on the gel are amplified25S rRNA from Candida (yeast that can cause sepsis). The column headingsare −Ctl which denotes a benchtop negative control. +Ctl is the benchtoppositive control. S1-S4 are four CARD runs. The white box results foreach S1-S4 are from an aliquot removed from the CARD and amplified priorto the second step of the purification. In this embodiment, the nucleicacid was first purified, then eluted (there were 2 stages to thepurification and 2 elutions: one from the first stage to the secondstage and then elution from the second stage) then amplified, then runon a gel. The results confirm that the second stage of the purificationprovides a marked improvement of the target's amplification. In thiscase, the primer amplified the 25S rRNA from Candida which was spikedinto a whole blood sample.

Referring to FIG. 24, rather than blending the specimen as is typicallydone, the tissue sample is dissected with a 3 mm diameter cutter. Thecutter is then inserted into a sample reservoir whereupon operating thecutter plunger, the dissected sample is transferred to the analysischamber. More specifically, with reference to FIG. 25, the sample isloaded as described immediately above; a controller dispenses lysisbuffer/proteinase K into the reagent reservoir; the lysisbuffer/proteinase K is pumped into the sample reservoir; the mixture iscirculated; the controller dispenses ethanol into the reagent reservoir;ethanol is mixed with the reaction solution; the reaction solution ispumped into the purifier on top of the silica filter; and the reactionsolution is pumped through the silica filter into the waste reservoirvia the silica membrane. Additionally, a screen may be inserted into thereservoir prior to the insertion of the sample so that the tissue passesthrough the screen in order to increase the surface area of the specimenfor the reagents to react more efficiently with the specimen.

FIG. 22 illustrates an exploded view of a double assay unit arrangementof a channel style magnetic separation/concentration system showing thereservoir layer 17, the fluidic transport layer 16, the film layer 30,and the out of plane amplification reactors 31 showing their componentparts. FIG. 22 also includes the silica filter system 36 a and 36 b.

FIG. 46 illustrates an alternative version of a self-containedbiological assay apparatus 10 including an optical system 60 for theperformance of real-time PCR analysis of nucleic acid amplificationreactions (for clarity certain elements of the dispensing system 13 andthe camera 27 have been removed) according to an embodiment of theinvention.

FIG. 47 illustrates schematic view of an optical system 60 suited togenerate a wide spectrum of source light 63 that is directed intoamplification reactor 31 through optical channel 66 formed into thereservoir layer 17 above amplification reactor 31. The source lightcauses fluorescent emission from certain known reagents in theamplification reactor 31. The fluorescent emission is then separatedthrough a series of dichroic mirrors 65 into each of a particularphotodiode 62. The resulting signal obtained from each particularphotodiode 62 during each particular thermocycle during the PCR reactionis recorded and data later analyzed in accordance with generally knownreal-time PCR methods.

FIG. 48a illustrates the internal components of a single light source 63single detector 62 alternative arrangement of an optical system 60. Thealternative arrangement therefore requires more than one componentassembled together to become the optical system 60. FIG. 46 shows fouroptical system components 60 assembled onto dispensing system 13, moreor fewer optical system components 60 may be assembled onto thedispensing system 13 for exciting more or fewer particular fluorescententities in the expected to be used in a real-time PCR reaction.

FIG. 48b illustrates a schematic diagram for using the alternativeoptical system illustrated in FIG. 48a . Using the alternative systemrequires multiple optical systems installed on the instrument—one foreach fluorophore expected to be used in a real-time PCR reaction. Theoperation of the alternative system illustrated in FIG. 48a is similarto the operation of the optical system illustrated in FIG. 47 wherebythe light from the optical system is directed into amplification reactor31 through optical channel 66 formed into the reservoir layer 17 aboveamplification reactor 31. Each light source in turn causes fluorescentemission from certain known reagents in the amplification reactor 31.The fluorescent emission is then directed by a dichroic mirror 65 intoits particular photodiode 62. The resulting signal obtained from eachparticular photodiode 62 during each particular thermocycle during thePCR reaction is recorded and data later analyzed in accordance withgenerally known real-time PCR methods.

FIG. 49 illustrates a layered top plan view of a “four assay unit”arrangement of the reservoirs 17, optical channels 66 and amplificationreactors 31. In operation during the thermalcycling stage of an assaythe optical system 60 is moved over each optical channel 66 and thelight generated by the light source 63 is directed through the opticalchannel 66 into amplification reactor 31. The light is absorbed by afluorophore in accordance with generally know real-time PCR systems andthen the resulting emitted light is directed back out through theoptical channel 66 back into the optical system 60 where it is detectedby photodiode 62 and the resulting data is analyzed using generallyknown real-time PCR analysis. The process is repeated for each reactoreach thermocycle until the reaction is complete.

FIG. 50 illustrates a perspective view of a “four assay unit”arrangement of the reservoirs 17, optical channels 66 and amplificationreactors 31. In operation during the thermalcycling stage of an assaythe optical system 60 is moved over each optical channel 66 and thelight generated by the light source 63 is directed through the opticalchannel 66 into amplification reactor 31. The light is absorbed by afluorophore in accordance with generally known real-time PCR systems andthen the resulting emitted light is directed back out through theoptical channel 66 back into the optical system 60 where it is detectedby photodiode 62 and the resulting data is analyzed using generallyknown real-time PCR analysis. The process is repeated for each reactoreach thermocycle until the reaction is complete.

FIG. 51 illustrates a cross sectional view of a “four assay unit”arrangement of the reservoirs 17, fluidic transport layer 16, opticalchannels 66 and amplification reactors 31. In operation during thethermalcycling stage of an assay the optical system 60 is moved overeach optical channel 66 and the light generated by the light source 63is directed through the optical channel 66 into amplification reactor31. The light is absorbed by a fluorophore in accordance with generallyknown real-time PCR systems and then the resulting emitted light isdirected back out through the optical channel 66 back into the opticalsystem 60 where it is detected by photodiode 62 and the resulting datais analyzed using generally known real-time PCR analysis. The process isrepeated for each reactor each thermocycle until the reaction iscomplete.

FIG. 52a illustrates an exploded view of a “four assay unit” arrangementof the reservoirs 17, fluidic transport layer 16, optical channels 66and amplification reactors 31. In operation during the thermalcyclingstage of an assay the optical system 60 is moved over each opticalchannel 66 and the light generated by the light source 63 is directedthrough the optical channel 66 into amplification reactor 31. The lightis absorbed by a fluorophore in accordance with generally knownreal-time PCR systems and then the resulting emitted light is directedback out through the optical channel 66 back into the optical system 60where it is detected by photodiode 62 and the resulting data is analyzedusing generally known real-time PCR analysis. The process is repeatedfor each reactor each thermocycle until the reaction is complete.

FIG. 52b illustrates an exploded view of an alternative construction ofa “four assay unit” arrangement where the reservoirs 17 and the fluidictransport layer 16 are embodied in the same substrate as opposed to twoseparate substrates, optical channels 66, film layer 30 andamplification reactors 31 In operation during the thermalcycling stageof an assay the optical system 60 is moved over each optical channel 66and the light generated by the light source 63 is directed through theoptical channel 66 into amplification reactor 31. The light is absorbedby a fluorophore in accordance with generally known real-time PCRsystems and then the resulting emitted light is directed back outthrough the optical channel 66 back into the optical system 60 where itis detected by photodiode 62 and the resulting data is analyzed usinggenerally known real-time PCR analysis. The process is repeated for eachreactor each thermocycle until the reaction is complete.

Nucleic Acids

In certain embodiments, the invention provides a method of amplifyingand/or isolating nucleic acid molecules of interest (also referred toherein as “nucleic acids of interest,” “target nucleic acids,” “targetpolynucleotides”). An isolated nucleic acid molecule (or “isolatednucleic acid”) is a nucleic acid molecule (or “nucleic acid”) that isseparated from other nucleic acid molecules that are present in thenatural source of the nucleic acid molecule. Preferably, an “isolated”nucleic acid is free of nucleic acid sequences (e.g., protein encodingsequences) that naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. In otherembodiments, the isolated nucleic acid is free of intron sequences.

“Nucleic acids of interest,” “target nucleic acids” or “targetpolynucleotides” refer to molecules of a particular polynucleotidesequence of interest. Such nucleic acids of interest that may beanalyzed by the methods of the present invention include, but are notlimited to DNA molecules such as genomic DNA molecules, cDNA moleculesand fragments thereof, including oligonucleotides, expressed sequencetags (“ESTs”), sequence tag sites (“STSs”), etc. Nucleic acids ofinterest that may be analyzed by the methods of the invention alsoinclude RNA molecules such as, but by no means limited to messenger RNA(mRNA) molecules, ribosomal RNA (rRNA) molecules, cRNA (i.e., RNAmolecules prepared from cDNA molecules that are transcribed in vivo) andfragments thereof. In various embodiments, the isolated nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb or 0.1 kb of nucleotide sequences that naturally flank the nucleicacid molecule in genomic DNA of the cell from which the nucleic acid isderived. Moreover, an isolated nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material, ofculture medium when produced by recombinant techniques, or of chemicalprecursors or other chemicals when chemically synthesized.

The nucleic acids of interest can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof. The nucleic acid can bemodified at the base moiety, sugar moiety, or phosphate backbone, andmay include other appending groups or labels.

For example, in some embodiments the nucleic acid can comprise at leastone modified base moiety which is selected from the group including butnot limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4 acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

In another embodiment, the nucleic acid can comprise at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the nucleic acid can comprise at least onemodified phosphate backbone selected from the group including but notlimited to a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

Nucleic acids for use as primers, probes, or templates may be obtainedcommercially or derived by standard methods known in the art, e.g., byuse of an automated DNA synthesizer (such as those commerciallyavailable from Biosearch Technologies, Inc., Novato, Calif.; AppliedBiosystems, Foster City, Calif., etc.) and standard phosphoramiditechemistry; or by cleavage of a larger nucleic acid fragment usingnon-specific nucleic acid cleaving chemicals or enzymes or site-specificrestriction endonucleases.

If the sequence of a nucleic acid of interest from one species is knownand the counterpart gene from another species is desired, it is routinein the art to design probes based upon the known sequence. The probeshybridize to nucleic acids from the species from which the sequence isdesired, for example, hybridization to nucleic acids from genomic or DNAlibraries from the species of interest.

In one embodiment, a nucleic acid molecule is used as a probe that iscomplementary to, or hybridizable under moderately stringent conditionsto, an amplified, isolated nucleic acid of interest.

In another embodiment, a nucleic acid molecule is used as a probe thathybridizes under moderately stringent conditions to, and is at least 95%complementary to, an amplified nucleic acid of interest.

In another embodiment, a nucleic acid molecule is used as a probe thatis at least 45% (or 55%, 65%, 75%, 85%, 95%, 98%, or 99%) identical to anucleotide sequence of interest or a complement thereof.

In another embodiment, a nucleic acid molecule is used as a probe thatcomprises a fragment of at least 25 (50, 75, 100, 125, 150, 175, 200,225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650,700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2400, 2600, 2800,3000, 3200, 3400, 3600, 3800, or 4000) nucleotides of a nucleic acid ofinterest or a complement thereof.

In another embodiment, a nucleic acid molecule is used as a probe thathybridizes under moderately stringent conditions to an amplified nucleicacid molecule having a nucleotide sequence of interest, or a complementthereof. In other embodiments, a nucleic acid molecule is used as aprobe that can be at least 25, 50, 75, 100, 125, 150, 175, 200, 225,250, 275, 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700,800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800,3000, 3200, 3400, 3600, 3800, or 4000 nucleotides in length andhybridize under moderately stringent conditions to an amplified nucleicacid molecule of interest or a complement thereof.

Nucleic acids that can be used as probes (or templates) for detecting anamplified nucleic acid of interest can be obtained by any method knownin the art, e.g., from a plasmid, by polymerase chain reaction (PCR)using synthetic primers hybridizable to the 3′ and 5′ ends of thenucleotide sequence of interest and/or by cloning from a cDNA or genomiclibrary using an oligonucleotide probe specific for the nucleotidesequence. Genomic clones can be identified by probing a genomic DNAlibrary under appropriate hybridization conditions, e.g., highstringency conditions, low stringency conditions or moderate stringencyconditions, depending on the relatedness of the probe to the genomic DNAbeing probed. For example, if the probe for the nucleotide sequence ofinterest and the genomic DNA are from the same species, then highstringency hybridization conditions may be used; however, if the probeand the genomic DNA are from different species, then low stringencyhybridization conditions may be used. High, low and moderate stringencyconditions are all well known in the art.

Amplified nucleic acids of interest can be detectably labeled usingstandard methods known in the art.

The detectable label can be a fluorescent label, e.g., by incorporationof nucleotide analogs. Other labels suitable for use in the presentinvention include, but are not limited to, biotin, imminobiotin,antigens, cofactors, dinitrophenol, lipoic acid, olefinic compounds,detectable polypeptides, electron rich molecules, enzymes capable ofgenerating a detectable signal by action upon a substrate, andradioactive isotopes. Preferred radioactive isotopes include, 32P, 35S,14C, 15N and 125I, to name a few. Fluorescent molecules suitable for thepresent invention include, but are not limited to, fluorescein and itsderivatives, rhodamine and its derivatives, texas red,5′-carboxy-fluorescein (“FMA”),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxy-fluorescein (“JOE”),N,N,N′,N′-tetramethyl-6-carboxy-rhodamine (“TAMRA”),6′-carboxy-X-rhodamine (“ROX”), HEX, TET, IRD40 and IRD41. Fluorescentmolecules that are suitable for the invention further include: cyaminedyes, including but not limited to Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 andFluorX; BODIPY dyes, including but not limited to BODIPY-FL, BODIPY-TR,BODIPY-TMR, BODIPY-630/650, and BODIPY-650/670; and ALEXA dyes,including but not limited to ALEXA-488, ALEXA-532, ALEXA-546, ALEXA-568,and ALEXA-594; as well as other fluorescent dyes known to those skilledin the art. Electron rich indicator molecules suitable for the presentinvention include, but are not limited to, aferritin, hemocyanin, andcolloidal gold. Alternatively, an amplified nucleic acid of interest(target polynucleotide) may be labeled by specifically complexing afirst group to it. A second group, covalently linked to an indicatormolecule and which has an affinity for the first group, can be used toindirectly detect the target polynucleotide. In such an embodiment,compounds suitable for use as a first group include, but are not limitedto, biotin and iminobiotin.

The nucleic acids of interest that are amplified and analyzed (e.g.,detected) by the methods of the invention can be contacted to a probe orto a plurality of probes under conditions such that polynucleotidemolecules having sequences complementary to the probe hybridize thereto.As used herein, a “probe” refers to polynucleotide molecules of aparticular sequence to which nucleic acid molecules of interest having aparticular sequence (generally a sequence complementary to the probesequence) are capable of hybridizing so that hybridization of the targetpolynucleotide molecules to the probe can be detected. Thepolynucleotide sequences of the probes may be, e.g., DNA sequences, RNAsequences or sequences of a copolymer of DNA and RNA. For example, thepolynucleotide sequences of the probes may be full or partial sequencesof genomic DNA, cDNA, mRNA or cRNA sequences extracted from cells. Thepolynucleotide sequences of the probes may also be synthesized, e.g., byoligonucleotide synthesis techniques known to those skilled in the art.The probe sequences can also be synthesized enzymatically in vivo,enzymatically in vitro (e.g., by PCR) or non-enzymatically in vitro.

Preferably, the probes used in the methods of the present invention areimmobilized to a solid support or surface such that polynucleotidesequences that are not hybridized or bound to the probe or probes may bewashed off and removed without removing the probe or probes and anypolynucleotide sequence bound or hybridized thereto. Methods ofimmobilizing probes to solid supports or surfaces are well known in theart. In one particular embodiment, the probes will comprise an array ofdistinct polynucleotide sequences bound to a solid (or semi-solid)support or surface such as a glass surface or a nylon or nitrocellulosemembrane. Most preferably, the array is an addressable array whereineach different probe is located at a specific known location on thesupport or surface such that the identity of a particular probe can bedetermined from its location on the support or surface. In a specificembodiment, the method described in WO 2009/049268 A1 by Zhou et al.(published Apr. 16, 2009) can be used to immobilize nucleic acid probesto a solid support or surface.

Although the probes used in the invention can comprise any type ofpolynucleotide, in preferred embodiments the probes compriseoligonucleotide sequences (i.e., polynucleotide sequences that arebetween about 4 and about 200 bases in length, and are more preferablybetween about 15 and about 150 bases in length). In one embodiment,shorter oligonucleotide sequences are used that are between about 4 andabout 40 bases in length, and are more preferably between about 15 andabout 30 bases in length. However, a more preferred embodiment of theinvention uses longer oligonucleotide probes that are between about 40and about 80 bases in length, with oligonucleotide sequences betweenabout 50 and about 70 bases in length (e.g., oligonucleotide sequencesof about 60 bases in length) being particularly preferred.

Uses of the CARD

It will be apparent to the skilled artisan that CARD-based diagnosticassays, as disclosed herein, can be used for many different applicationsin which bench-top based assays are currently used. The design of theplastic CARD permits the incorporation of all necessary microfluidicnetworks, valves, pumps and reservoirs on a simple, inexpensivedisposable microfluidic device. Since all assay functions (i.e., flowand mixing rates, temperature control, including thermocycling, residenttimes, etc.) are easily controlled by software, sophisticated multiplexPCR assays can be easily performed by individuals of varying skilllevel. Furthermore, CARDs can be inserted in either a portable, batteryoperated POC controller or into a higher throughput EncompassMDx™workstation. Regardless of the format selected, however,ease-of-performance is achieved.

The CARD can be adapted by the skilled artisan to assay for any nucleicacid sequence of interest through choice of primer for amplification andchoice of probe for detection.

In one embodiment, the CARD can be used for conducting moleculardiagnostics assays, which can provide a basis for the management ofpotential disease states based upon an individual's genomic background.

In another embodiment, the CARD can be used for conducting screens forpharmacogenomic sensitivity, e.g., genetic predisposition forsensitivity to a drug, pharmaceutical composition, chemical or compoundof interest.

In another embodiment, the CARD can be used for conducting oncogenicscreening assays, i.e., screening for a nucleic acid of interest that isassociated with predisposition for cancer.

In another embodiment, the CARD can be used for conducting screeningassays for infectious disease agents, pathogens or sepsis.

In another embodiment, the CARD can be used for analysis of singlenucleotide polymorphisms (SNPs) for oncology purposes, pharmacogenomicpurposes, companion diagnostics (dosing or other needs specific to aparticular pharmaceutical compound) or to detect communicable ornoncommunicable infectious diseases.

In another embodiment, the CARD can be used for industrial orenvironmental assays for organisms of interest that are infectious tohumans, animals or plants or for spoilage organisms in processed foodsor non-processed foods.

In another embodiment, the CARD can be used industrially for conductingassays for monitoring recreational water (beaches, pools water parks,etc.) or water treatment systems for drinking water, ballast water ortreated waste water.

In another embodiment, the CARD can be used for conducing screeningassays for sparse target nucleic acids distributed in a large volume ofliquid.

In another embodiment, the CARD can be used for conducing screeningassays for sparse target nucleic acids wherein the sparse targets are tobe distinguished from a high background of non-target nucleic acids in asample.

In certain embodiments, the CARD can be easily adapted to have multipleamplification reactors (e.g., 31, FIGS. 11, 12) that sequester competingprimers and that feed into a single analysis reservoir 41, so thatmultiple amplification reactions can be conducted concurrently onnucleic acids from a single sample.

The CARD can be adapted by the skilled artisan to perform any thermallymediated nucleic acid amplification known in the art including but notlimited to: polymerase chain reaction (PCR), reverse-transcriptase (RT-)PCR, Rapid Amplification of cDNA Ends (RACE), rolling circleamplification, Nucleic Acid Sequence Based Amplification (NASBA),Transcript Mediated Amplification (TMA), Ligase Chain Reaction,transcription-associated amplification (TAA), Cold PCR and non-enzymaticamplification technology. (NEAT).

In addition to heating the CARD for nucleic acid amplification, the CARDcan be heated to test for viability of a detected organism (e.g., thepresence of heat-shock associated RNA expressed by the organism).Heating can also be used to regulate the stringency of hybridization inanalyses such as the detection of single nucleotide polymorphisms(SNPs).

The CARD can be adapted by the skilled artisan to accommodate anyanalytic or detection method for amplicons known in the art, includingbut not limited to: colorimetric, fluorescent colorimetric,chemiluminescence, electrochemical, electrophoretic, lateral flow,protein microarray, nucleic acid microarray, fluorescence detectionmethods or various combinations of the detection methods listed above.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Pharmacogenomic Assay for Warfarin Sensitivity

This example demonstrates a specific embodiment of a pharmacogenomicassay for warfarin sensitivity that has been fully integrated andautomated on the CARD. Reverse dot blot (RDB) was conducted to analyzethe results, although as indicated below, the primer extension methodcan also be used. The protocol described below can be easily adapted bythe skilled practitioner to assay for any single nucleotide polymorphism(SNP) of interest through choice of primers and probes, and can be usedfor other assays, e.g., oncogenic screening assays.

In general, assays conducted on the CARD comprise the following steps:

1. Direct application of a raw specimen to the CARD (the only operatorstep).

2. Chemical cell lysis.

3. Nucleic acid extraction and purification via binding to a silicacolumn contained in the CARD.

4. Elution of purified nucleic acids from the silica column.

5. Mixing of an aliquot of the purified nucleic acids with a PCR mastermixture containing all of the reagents needed to perform PCRamplification including buffer, primers, nucleotide triphosphates,magnesium chloride, Taq DNA polymerase and Uracil-DNA Glycosylase, whichis used to insure against the unlikely event of amplicon cross-overcontamination.6. Introduction of the complete mixture into the PCR thermocyclerchambers located directly above the resistive heaters embedded in themanifold, followed by initiation of the thermocycling program.7. Following completion of the thermocycling program, introduction ofthe amplicons into the detection module where they are subjected to anassay, e.g., by the primer extension method (FIG. 26A-B) or a reversedot blot (RDB) assay (FIG. 29).8. Imaging and analysis of the detected spots on analysis membrane,e.g., via the primer extension method or RDB membrane.9. Objective results provided to the user.

Although PCR amplification is employed in this embodiment, any thermallymediated nucleic acid amplification known in the art can be performedusing the above approach, including but not limited to: polymerase chainreaction (PCR), reverse-transcriptase (RT-) PCR, Rapid Amplification ofcDNA Ends (RACE), rolling circle amplification, Nucleic Acid SequenceBased Amplification (NASBA), Transcript Mediated Amplification (TMA),Ligase Chain Reaction, transcription-associated amplification (TAA),Cold PCR and non-enzymatic amplification technology. (NEAT).

In reference to FIGS. 14A-14L and the reservoir variation in FIG. 18,the following operations and reagents were used to performpharmacogenomic studies pertaining to single nucleotide polymorphisms(“SNPs”) of three individual SNPs known to be indicators of warfarinsensitivity: CYP2C9*2, CYP2C9*3, and VKORC1*2.

Probe Set

The following set of probes was used for detecting CYP2C9*2, CYP2C9*3,and VKORC1*2:

CYP2C9*2 WT/5AmMC6/TGAGGACCGTGTTCA (SEQ ID NO:113)

CYP2C9*2 MUT/5AmMC6/TGAGGACTGTGTTCA (SEQ ID NO:114)

CYP2C9*3 WT/5AmMC6/A AGG TCA ATG TAT CTC T (SEQ ID NO:115)

CYP2C9*3 MUT/5AmMC6/AGG TCA AGG TAT CTC (SEQ ID NO:116)

VKORC1_WT/5AmMC6/CAT CGA CCC TTG GAC (SEQ ID NO:117)

VKORC1_MUT/5AmMC6/GTC CAA GAG TCG ATG A (SEQ ID NO:118)

Sample Addition and Cell Lysis

a. An operator adds a 5 μl sample of either blood or suspended buccalswabs into the sample input reservoir.

b. Dispense 30 μl of a cell storage buffer into the reagent inputreservoir and pump it into the sample input reservoir.

c. Dispense 30 μl of a mixture of Proteinase K and lysis buffer into thereagent input reservoir and pump it into the sample input reservoir andincubate for 5 minutes.

d. Dispense 30 μl of ethanol into the reagent input reservoir and pumpit into the sample input reservoir.

e. Pump the entire contents of the sample input reservoir on top of thefilter in the purification reservoir and then pull the contents throughthe filter and pump it to waste.

f. Dispense 40 μl of ethanol into the reagent input reservoir and pumpit onto the top of the filter in the purification reservoir and thenpull the contents through the filter and pump it to waste.

g. Dispense 70 μl of wash buffer 1 into the reagent input reservoir andpump it onto the top of the filter in the purification reservoir andthen pull the contents through the filter and pump it to waste.

h. Dispense 70 μl of wash buffer 2 into the reagent input reservoir andpump it onto the top of the filter in the purification reservoir andthen pull the contents through the filter and pump it to waste.

i. Dispense 90 μl of water into the reagent input reservoir and pump itto waste.

j. Dispense 70 μl of elution buffer into the reagent input reservoir andpump it onto the top of the filter in the purification reservoir andthen pump it to waste back through the channel it came from. Then pullany remaining fluid through the filter and pump it to waste.

Elution

a. Dispense 50 μl of elution buffer into one of the elution reservoirsand pump a portion of it to the other elution reservoir then pump theremainder up through the bottom of the filter in the purificationreservoir and then fluff it by alternatively filling and emptying thepurification reservoir at least 2 times.

PCR Loading

a. Dispense 12 μl of PCR master mix 1 into one of the master mixreservoirs and then repeat with 12 μl of PCR master mix 2 into the othermaster mix reservoir.

b. Pump a small amount (3 flexes of the diaphragm) of the contents ofthe purification reservoir to the second elution reservoir to whichelution buffer was pumped.

c. Pump a single flex of the diaphragm to move a small amount of thematerial from the purification reservoir to one of amplificationreservoirs then fill that same amplification reservoir with the mastermix from the master mix reservoir on the same side as the amplificationreservoir. Repeat to fill the second amplification reservoir.

PCR

a. 10 minutes at 37° C.

b. 2 minutes at 95° C.

c. Cycle 40×

i. 30 seconds at 95° C.

ii. 30 seconds at 45° C.

iii. 30 seconds at 72° C.

iv. 3 minutes at 72° C.

Reverse Dot Blot (RDB) Filter Blocking

RDB filter blocking is performed during thermocycling so that the twosteps are coordinated with the completion of the amplification.

a. Dispense 80 μl of water into the analysis reagent reservoir and pumpit to the analysis filter and then circulate alternatively clockwise andcounterclockwise 15 times each to waste.

b. Dispense 80 μl of 0.1N NaOH into the analysis reagent reservoir andpump it to the analysis filter and circulate it alternatively clockwiseand counterclockwise 15 times and then pump to waste.

c. Dispense 80 μl of water into the analysis reagent reservoir and pumpit to the analysis filter and then circulate alternatively clockwise andcounterclockwise 15 times each to waste. Repeat this step 2 more times.

Pre-Hybridization

a. Dispense 80 μl of SS buffer (0.15M NaCl+0.01M Sodium Phosphate+0.001MEDTA+0.1% SDS with final pH 7.25-7.50) into the analysis reagentreservoir and pump it to the analysis filter and circulate it Dispense80 μl of water into the analysis reagent reservoir and pump it to theanalysis filter and then circulate alternatively clockwise andcounterclockwise 5 times and then incubate without circulation for 10minutes.

Amplicon Withdrawal

During the 10 minute incubation for pre-hybridization, these processesmay begin.

a. Increase temperature in the amplification reactors to 95° C. for 30seconds just prior to adding the SS buffer.

b. Concurrently dispense 80 □l of SS buffer into the analysis reagentreservoir and pump it into one of the amplification reactors. Repeat forthe other amplification reactor. Incubate each amplification reactor for3 minutes.

c. Turn off the amplification heaters and cool the amplificationreactors allowing the wax/silicone to harden the sealing layer so thatwhen removing the amplicons the liquid phase wax/silicone layer is notremoved with the amplicons.

Hybridization

a. Concurrently with the amplicon withdrawal steps a-c, empty theanalysis reservoir and increase the temperature of the heater under theanalysis reservoir to 50° C. to heat the membrane.

b. Pump 3 strokes each for each amplification reactor to pump some ofthe contents of each of the reactors to the analysis membrane.

c. Incubate by circulating alternatively clockwise and counterclockwisethe contents of the analysis reservoir for fifteen minutes. It is bestto use the covered analysis reservoir variation to improve the reaction.Then empty the contents to waste.

d. Dispense 80 μl of SS buffer into the analysis reagent reservoir andpump it to the analysis filter and then circulate alternativelyclockwise and counterclockwise 15 times each then pump to waste. Repeat2 times.

e. Decrease the temperature of the analysis reservoir to less than 30°C.

f. Dispense 80 μl of SS buffer into the analysis reagent reservoir andpump it to the analysis filter and then circulate it once then incubateit for 5 minutes circulating it once per minute and then pump it towaste.

Conjugation

a. Dispense 80 μl of HRP into the analysis reagent reservoir and pump itto the analysis membrane circulate it alternatively clockwise andcounterclockwise for 10 minutes then pump to waste.

b. Dispense 80 μl of SS buffer into the analysis reagent reservoir andpump it to the analysis filter and then circulate alternativelyclockwise and counterclockwise 15 times each then pump to waste. Repeat3 times.

Substrate Addition

a. Dispense 80 μl of TMB into the analysis reagent reservoir and pump itto the analysis membrane circulate alternatively clockwise andcounterclockwise for 5 minutes then pump to waste.

b. Dispense 80 μl of water into the analysis reagent reservoir and pumpit to the analysis membrane circulate alternatively clockwise andcounterclockwise 15 times then pump to waste. Repeat 2 times.

Image Analysis

a. Position the camera over the analysis membrane and record the image.

b. Send the image to the control system for processing.

c. Report the results.

Example 2 CARD-Based Methods for Rapid and Automatic Detection of SingleNucleotide Polymorphisms (SNPs)

Introduction

This example demonstrates use of the CARD for the evolving moleculardiagnostics industry that incorporates low cost, CARD technology toanalyze clinical raw samples. Once a raw specimen is introduced into theCARD, all assay functions, including cell lysis, nucleic acidpurification, multiplex PCR, and end-point analysis, are automaticallyperformed.

The CARD was used in a pharmacogenomic assay to detect single nucleotidepolymorphisms (SNPs) associated with warfarin sensitivity. Raw buccalswab samples from twenty individual volunteers were analyzed and the SNPprofiles, identified by the warfarin sensitivity assay carried out onthe CARD, were confirmed via bi-directional DNA sequencing. Thepharmacogenomics protocol described below, however, can be easilyadapted by the skilled practitioner to assay for any nucleic acidsequence or SNP of interest through choice of primers and probes. Suchan assay could be used, for example, to screen for viral pathogens, foroncogenes or other genetic mutations, variants or markers of interest,and virtually any cell or tissue can be assayed.

Background

The use of molecular diagnostics has expanded greatly since itsinception in the early 1980s, particularly as a means to permit thedetection of slow growing or fastidious bacteria responsible forinfectious diseases. The detection of viral pathogens, including viralload testing has also been significantly improved by moleculardiagnostics. As more data have become available regarding the humangenome, the use of molecular diagnostics in pharmacogenomic, companiondiagnostics, and other personalized medicine applications continues togain momentum. Despite its power and versatility, however, the need forhighly trained personnel and expensive capital equipment has restrictedthe use of molecular diagnostics to specialized laboratories or centrallabs suitably equipped and staffed.

While many effective “point-of-care” (POC) diagnostics have beendeveloped that rely upon immunological assays in a lateral flow assayformat (e.g., pregnancy tests), the ability to perform the more complexmolecular assays has not yet been fully achieved in an easy-to-use andinexpensive POC format. Before molecular diagnostics can be more broadlyused in various POC settings, the assays need to be simplified andequipment requirements reduced. Currently, “bench top” molecular assaysrequire significant effort by highly trained personnel to prepare thesamples for analysis, starting with raw clinical specimens.Subsequently, the gene amplification and detection steps also requiresignificant skill and expensive equipment. Moreover, while lateral flowPOC assays frequently rely upon subjective interpretation of colorintensity on test strips, results from more sophisticated molecularassays would be more meaningful if unambiguous and objective digitalresults can be provided. If all of these processes could be integratedin a seamless, fully automated manner, individuals of varying skilllevel could perform a range of POC molecular assays and achieveobjective, clear-cut interpretation of results in an economical assayformat.

The challenges posed by the molecular POC markets have led to theintroduction of several “sample-to-results” platforms, but most stillrequire either separate “sample preparation” steps and/or equipment orconsiderable “pre-preparation” of the sample prior to introduction intothe system to achieve gene amplification and detection. To achieve true“sample-to-results” simplicity a platform has been developed thatintegrates all required sample preparation, assay, and detection stepsinto a single, inexpensive disposable plastic device capable ofachieving fully automated molecular diagnostic testing. The CARDdemonstrated in this example requires only the introduction of a rawspecimen, with all subsequent steps performed automatically. Thedevice's low cost of both capital equipment and disposables, as well asthe absence of any “hands on” efforts, will help make moleculardiagnostics a reality in the entire spectrum of critical andpoint-of-care testing.

Materials and Methods

Assays

“Bench top” assays were optimized to establish various parameters thatwere then converted to the fully automated platform of the CARD. Primersand probes were designed, using methods known in the art, foramplification and capture, respectively. This could also involveoptimization of standard chemical lysis and nucleic acid purificationprotocol if any of the organisms being analyzed were too tough to belysed.

All sequences were obtained from the National Center for Biotechnology(NCBI) information (www.ncbi.nlm.nih.gov). Primers and probes weredesigned using CLC Sequence Viewer (www.clcbio.com), Integrated DNATechnologies SciTools, (www.idtdna.com/scitools/) and NCBI Primer Blast(www.ncbi.nlm.nih.gov/tools/primer-blast) using standard methods knownin the art. All primers and probes were synthesized at Integrated DNATechnologies (Coralville, Iowa). All microbial and viral DNAs werepurchased from the American Type Culture Collection (ATCC, Manassas,Va.).

Warfarin Sensitivity Assay

De-identified buccal swabs were obtained from volunteers followinginformed consent and cells were lysed and DNA extracted. DNA wassubjected to amplification on the CARD using primers designed to amplifythe regions surrounding three individual SNPs known to inform warfarinsensitivity CYP2C9*2, CYP2C9*3, and VKORC1*2 (27-32). The CYP2C9*2 andCYP2C9*3 SNPs correspond to mutations in the cytochrome P450 gene, andthe VKORC1*2 corresponds to a mutation in the vitamin K epoxidereductase complex subunit 1 gene.

Following purification, the DNA was separated into two distinct PCRreactions; one mix contained primers to amplify the regions surroundingboth CYP2C9 mutations and the other mix contained primers to amplify theregion surrounding the VKORC1*2 mutation. Following PCR, the ampliconsfrom both reaction mixes were mixed, denatured, and then moved to achamber containing probes covalently linked to the membrane filters. Thedenatured amplicons were annealed to the capture probes in the presenceof buffer, dNTPs including biotinylated dUTP, magnesium ions, and DNApolymerase lacking the 3′-5′ exonuclease proof-reading function (e.g.,Vent Polymerase, New England Biolabs, Ipswich, Mass.).

For the primer extension assay, the immobilized probes, approximately 20nucleotides long, contained the informative nucleotide at their 3′termini. Under these conditions the denatured and annealed ampliconstrand behaves as the template, while the solid-phase probe representsthe “primer” to be extended. When an exact match is present between thetemplate and the primer, DNA synthesis occurs incorporating dNTPsincluding biotinylated dUTP. However, if there is a single mismatchbetween the terminal base of the immobilized “primer” and the template,elongation, and thus biotin incorporation does not occur (FIGS. 26A-B).

As conducted on the CARD, the thermocycling step of the primer extensionassay is performed in the analysis reservoir 41 (see FIGS. 14A-C).

Following one round of elongation, extended products were then detectedfollowing incubation with streptavidin conjugated HRP and TMB substrate.

Digitally captured images were subjected to analysis with Image Jsoftware (rsb.info.nih.gov/ij). The mean intensities of the spots weremeasured and the averages of the wild-type spots divided by the averageof the mutant probes. Ratios greater than, equal to, or less than 1correspond to a homozygous wild-type, heterozygous, or homozygous mutantgenotypes, respectively.

Confirmation of primer extended genotypes was achieved viabi-directional sequencing performed at the Cornell University LifeSciences Core Laboratories Center (Cornell University, Ithaca, N.Y.)using an Applied Biosystems Automated 3730DNA Analyzer with Big DyeTerminator chemistry (Rosenblum, B B, Lee, L G, Spurgeon, S L, et al.New dye-labeled terminators for improved DNA sequencing patterns.Nucleic Acid Res. 1997; 25: 4500-4504; Heiner, C R., Kunkapiller, K L,Chen, S-M., et al. Sequencing Multimegabase-Template DNA with BigDyeTerminator Chemistry. Genome Research 1998; 8: 557-561) and Ampli-Taq-FSDNA Polymerase (Applied Biosystems, Inc., Foster City, Calif.).

Results

This assay run on the CARD was designed to perform genotyping analysis(warfarin sensitivity assay) by identifying three separate SNPs known toinfluence the metabolism of warfarin. This single (VKORC1*2) andmultiplex (CYP2C9*2 and CYP2C9*3) PCR assays were designed to amplifythe regions surrounding each SNP and then the denatured amplicons weresubjected to primer extension assay to genotype each allele.

To evaluate the SNP assay on the CARD, buccal swabs from a total of 20volunteers were analyzed. Each sample was evaluated using (1) thewarfarin SNP assay run on the CARD and (2) bi-directional DNAsequencing. Using the primer extension assay, the warfarin sensitivityassay distinguishes between the various alleles found across the threedistinct warfarin-related SNPs. As shown in FIGS. 26A-B, PCR-amplifiedDNA sequences were denatured and allowed to hybridize to specificcapture probes immobilized on membrane filters. In this assay, thecapture probes ultimately served as the primers to be extended using theamplicons as templates. Incorporation of biotinylated dUTP along theprimer extended sequences results in streptavidinylated HRP binding andcolor detection as described in the Materials and Methods.

FIG. 27 is a representative image demonstrating how the genotypes wereread off of the primer extension filters, and FIG. 28 shows the 7different genotypes that were obtained from the 20 individuals. Thethree possible genotypes that exist for each of the three SNPsidentified can be detected by eye and/or established by determining theratio of signal intensity of the individual spots. When each of thebuccal swab samples from the 20 volunteers was evaluated on the warfarinsensitivity assay, the genotypes were originally read by at least twodifferent individuals. These results were confirmed with bi-directionalsequencing as well as with image analysis as described in Materials andMethods above.

The warfarin sensitivity assay in this example demonstrates theversatility of the CARD. Regardless of the assay(s) performed, only one“manual” (user conducted) step is required (i.e., initial introductionof the “raw” sample), with all subsequent steps computer automated.Owing to its ease of performance and interpretation of results, thisassay will be useful in POC settings in which physicians wish to usegenomic data, along with other clinical information, to help establishthe correct initial dosing of warfarin. Therefore instead of relyingupon time consuming and potentially dangerous “trial and error” dosingthat relies upon repetitive PT/INR testing to finally achieve propertherapeutic doses of warfarin, physicians could initially start warfarinat more appropriate doses, based upon several art-known warfarin dosingalgorithms (Daly, A K and King, B P. Pharmacogenetics of oralanticoagulants. Pharmacogenetics 2003; 13:247-252; Takahashi, H. andEschizen, H. Pharmacogenetics of warfarin elimination and its clinicalimplications. Clin. Pharmacokinet 2001; 40: 587-603; Schwarz, U I,Ritchie, M D, Bradford, Y, et al. Genetic determinants of response towarfarin during initial anticoagulation. N. Eng. J. Med 2008; 358:999-1008; Osinbowale, O., Al Malki, M., Schade, A., et al. An algorithmfor managing warfarin resistance. Clev. Clinic J. of Med. 2009; 76:724-730).

Assays run on the CARD provide a convenient, cost-effective means toperform sophisticated molecular assays in a completely “hands off”manner. Furthermore, the low capital costs of the equipment required torun the assays and the low disposable costs allows this platform tobring true “sample-to-results” molecular testing the point-of-caresettings.

Example 3 CARD-Based Automated Human Papilloma Virus (HPV) Assay

This example demonstrates a specific embodiment of a human papillomavirus (HPV) assay that has been fully integrated and automated on theCARD. The protocol described below can be easily adapted by the skilledpractitioner to assay for other nucleic acid sequences of interestthrough choice of primer for amplification and choice of probe for thearray.

A vaginal swab is collected in a suitable transport media, which allowsfor extended room temperature storage, if necessary. The transport mediacan be, e.g., PBS buffer, in which the sample will be immediatelyintroduced into the CARD for analysis. The transport media can also beis any type of solution known in the art that prevents DNA degradationso that the sample can be held for later use.

An aliquot is applied to the CARD and the run initiated as described inthe protocol below. Without any further intervention by the operator,all the following steps are automatically performed: cell lysis, nucleicacid purification, PCR amplification and multiplexed end-point detectionon a low density microarray. The following operations and reagents wereused to perform the HPV assay on the CARD. See FIGS. 14A-14L.

Sample Addition and Cell Lysis

a. Operator inserts a sample (e.g., a vaginal swab) into the sampleinput reservoir.

b. Dispense 30 μl of a mixture of Proteinase K and lysis buffer into thereagent input reservoir and pump it into the sample input reservoir andincubate for 5 minutes.

c. Dispense 30 μl of ethanol into the reagent input reservoir and pumpit into the sample input reservoir.

d. Pump the entire contents of the sample input reservoir on top of thefilter in the purification reservoir and then pull the contents throughthe filter and pump it to waste.

e. Dispense 40 μl of ethanol into the reagent input reservoir and pumpit onto the top of the filter in the purification reservoir and thenpull the contents through the filter and pump it to waste.

f. Dispense 70 μl of wash buffer 1 into the reagent input reservoir andpump it onto the top of the filter in the purification reservoir andthen pull the contents through the filter and pump it to waste.

g. Dispense 70 μl of wash buffer 2 into the reagent input reservoir andpump it onto the top of the filter in the purification reservoir andthen pull the contents through the filter and pump it to waste.

h. Dispense 90 μl of water into the reagent input reservoir and pump itto waste.

i. Dispense 70 μl of elution buffer into the reagent input reservoir andpump it onto the top of the filter in the purification reservoir andthen pump it to waste back through the channel it came from. Then pullany remaining fluid through the filter and pump it to waste.

Elution

a. Dispense 50 μl of elution buffer into one of the elution reservoirsand pump a portion of it to the other elution reservoir then pump theremainder up through the bottom of the filter in the purificationreservoir and then fluff it by alternatively filling and emptying thepurification reservoir at least 2 times.

Polymerase Chain Reaction (PCR) Loading

a. Dispense 12 μl of PCR master mix 1 into one of the master mixreservoirs and then repeat with 12 μl of PCR master mix 2 into the othermaster mix reservoir.

b. Pump a small amount (3 flexes of the diaphragm) of the contents ofthe purification reservoir to the second elution reservoir to whichelution buffer was pumped.

c. Pump a single flex of the diaphragm to move a small amount of thematerial from the purification reservoir to one of amplificationreservoirs then fill that same amplification reservoir with the mastermix from the master mix reservoir on the same side as the amplificationreservoir. Repeat to fill the second amplification reservoir.

Polymerase Chain Reaction (PCR)

a. 10 minutes at 37° C.

b. 2 minutes at 95° C.

c. Cycle 10×

i. 30 seconds at 95° C.

ii. 30 seconds at 46° C.

iii. 30 seconds at 72° C.

d. Cycle 30×

i. 15 seconds at 95° C.

ii. 30 seconds at 49° C.

iii. 30 seconds at 72° C.

iv. 3 minutes at 72° C.

Reverse Dot Blot (RDB) Filter Blocking

a. Begin reverse dot blot (RDB) filter blocking during the above PCRthermocycling so that the two steps are coordinated with the completionof the amplification.

b. Dispense 150 μl of 0.1N NaOH into the analysis reagent reservoir andpump it on top of the analysis filter and circulate it from the top ofthe membrane through the perforated ring and back to the top of themembrane 5 times and then pump to waste.

c. Dispense 90 μl of water into the analysis reagent reservoir and pumpit on top of the analysis filter and then circulate from the top of themembrane through the perforated ring and back to the top of the membrane3 times each then pump to waste. Repeat this step 1 more time.

Pre-Hybridization

a. Dispense 70 μl of hybridization buffer (0.15M NaCl+0.01M SodiumPhosphate+0.001M EDTA+0.1% SDS+15% formamide with final pH 7.25-7.50)into the analysis reagent reservoir and pump it on top of the analysisfilter and circulate it from the top of the membrane through theperforated ring and back to the top of the membrane 5 times then pump itto waste.

Amplicon Withdrawal

a. Dispense 70 μl of hybridization buffer into the analysis reagentreservoir and pump it onto the top of the analysis filter.

b. Dispense 70 μl of hybridization buffer into the analysis reagentreservoir and pump it into one of the amplification reactors. Repeat forthe other amplification reactor.

c. Turn off the amplification heaters and cool the amplificationreactors allowing the wax/silicone to harden the sealing layer so thatwhen removing the amplicons the liquid phase wax/silicone layer is notremoved with the amplicons.

Hybridization

a. Pump the contents of each amplification reactor to the top of theanalysis membrane.

b. Incubate by circulating (from the top of the analysis membranethrough the perforated ring and back to the top of the analysismembrane) the contents of the analysis reservoir for 12.5 minutes. Thenempty the contents to waste.

c. Dispense 90 μl of wash buffer into the analysis reagent reservoir andpump it to the top of the analysis filter and then circulate from thetop of the analysis membrane through the perforated ring and back to thetop of the analysis membrane 3 times each then pump to waste. Repeat 2times.

Conjugation

a. Dispense 120 μl of HRP into the analysis reagent reservoir and pumpit to the top of the analysis membrane. Circulate it from the top of theanalysis membrane through the perforated ring and back to the top of theanalysis membrane for 4 minutes then pump to waste.b. Dispense 90 μl of wash buffer into the analysis reagent reservoir andpump it to the top of the analysis filter. Then circulate from the topof the analysis membrane through the perforated ring and back to the topof the analysis membrane 3 times each then pump to waste. Repeat 3times.

Substrate Addition

a. Dispense 120 μl of TMB into the analysis reagent reservoir and pumpit to the top of the analysis membrane circulate from the top of theanalysis membrane through the perforated ring and back to the top of theanalysis membrane for 10 minutes then pump to waste.b. Dispense 90 μl of water into the analysis reagent reservoir and pumpit to the top of the analysis membrane. Then circulate from the top ofthe analysis membrane through the perforated ring and back to the top ofthe analysis membrane 15 times then pump to waste. Repeat 3 times.

Image Analysis

a. Position the camera over the analysis membrane and record the image.

b. Send the image to the control system for processing.

c. Report the results.

Example 4 CARD-Based Rapid Molecular Detection and Identification of 20Clinically Relevant HPV Types

This example demonstrates a method for rapidly, easily and automaticallydetecting and distinguishing at least 20 types of clinically relevanthuman papilloma virus (HPV) directly from clinical samples on the CARD,using the protocol discussed above in Example 3.

Introduction

Cervical cancer is the leading cause of cancer-related deaths amongwomen in low-income countries and is the second leading cause ofcancer-related deaths for women on a worldwide basis. Among currentlyFDA-approved molecular diagnostics, none are capable of distinguishingthe various HPVs other than to classify them as “high” or “low” risktypes.

Currently, two FDA approved molecular diagnostic tests are available inthe United States for the direct detection of HPV DNA. The HybridCapture test (Digene HC2, Qiagen, Valencia, Calif.) and the Cervista HPVtest (Hologic, Bedford Mass.). Both tests rely on signal amplificationrather than target amplification. However, both FDA approved kits do notidentify individual HR HPV, but rather the presence of a single ormultiple HR HPVs will be read as the same positive result. In addition,there is a hybrid capture kit for detecting LR HPVs as a group and aCervista kit specifically for the detection of only HPV 16 and 18.

Despite the availability of two highly predictive tests for determiningprobability of cervical cancer, the disparity of cervical cancermortality between low income and industrialized regions still remainssignificant. Cultural, socio-economic, and logistical barriers preventwomen in impoverished regions from benefiting from the predictive valueof these tests. The design of an inexpensive point-of-care device forthe molecular testing of HPV should significantly improve cervicalcancer detection worldwide. Such a test would provide immediate andunequivocal results regarding HPV status, and inform either the need forfurther treatment or the time to next check-up. The HPV nucleic acidtest demonstrated in this example is accessible to different populationsregardless of these aforementioned barriers.

Materials and Methods

Preparation, Dilution and Storage of Genomic DNA

The C-33A human cervical carcinoma cell line, purchased from theAmerican Type Culture Collection (ATCC, Manassas, Va.), was grown, andmaintained in Eagles Minimal Essential Media containing 10% fetal bovineserum at 37° C. in a CO2 water jacketed incubated. Cells were grown toconfluence, collected either via scraping directly into PBS, or throughtrypsinization followed by counting of cells. Approximately 5 millioncells (equivalent to 10 million genomes) were harvested and lysedfollowed by purification of genomic DNA using a Qiagen DNeasy kit(Qiagen, Valencia, Calif.). Concentration of nucleic acids wasdetermined via absorbance at 260 nm. Purified nucleic acids were storedat −20° C.

Preparation, Dilution and Storage of HPV plasmid DNA

Chimeric plasmid DNA containing HPV genomes were purchased from AmericanType Culture Collection (ATCC, Manassas, Va.) and transformed into DH5 αE. coli bacteria. Cultures were expanded, and plasmid DNA was purifiedusing Qiagen Plasmid Mini Kit (Qiagen, Valencia, Calif.). Concentrationof DNA was determined via absorbance at 260 nm, and DNA was diluted to1E6 copies per μl. Plasmid DNA stocks were stored at −20° C.

Preparation of DNA from Clinical Samples

All samples were originally collected as vaginal swabs in Digene storagetransport medium. A portion of the samples were also pre-treated withDigene denaturing solution. Two hundred μl of sample was subjected tonucleic acid purification with Qiagen DNAeasy or in-house purificationreagents which will be described elsewhere. Purified nucleic acid wasstored at −20° C.

Design of HPV Primers and Probes

The HPV L1 gene primer set was designed based on the HPV regioncorresponding to the 3′ end of the L2 gene and the 5′ end of the L1gene, originally described by Yoshikawa, and then further elaborated onby others (Yoshikawa H, Kawana T, Kitagawa K, Mizuno M, Yoshikura H,Iwamoto A: Detection and typing of multiple genital humanpapillomaviruses by DNA amplification with consensus primers. Jpn JCancer Res 1991, 82(5):524-531; Jeney C, Takacs T, Sebe A, Schaff Z:Detection and typing of 46 genital human papillomaviruses by the L1F/L1Rprimer system based multiplex PCR and hybridization. J Virol Methods2007, 140(1-2):32-42; Takacs T, Jeney C, Kovacs L, Mozes J, Benczik M,Sebe A: Molecular beacon-based real-time PCR method for detection of 15high-risk and 5 low-risk HPV types. J Virol Methods 2008,149(1):153-162). The sequence for each specific HPV type was obtainedfrom the National Center for Biotechnology information(www.ncbi.nlm.nih.gov). The HPV type and corresponding accession numberwere as follows: 6:NC_000904; 11:M14119; 16:NC_001526; 18:NC_001357;31:J04353; 33:M12732; 35:M74117; 39:M62849 M38185; 42:M73236;43:AJ620205; 44:U31788; 45:X74479; 51:M62877; 52:X74481; 53:NC_001593;56:EF177177; 58:D90400; 59:X77858; 66:U31794; 68:DQ080079.

Based on the primers described in Jeney et al. (Jeney C, Takacs T, SebeA, Schaff Z: Detection and typing of 46 genital human papillomavirusesby the L1F/L1R primer system based multiplex PCR and hybridization. JVirol Methods 2007, 140(1-2):32-42), the forward or reverse primerregion of the HPV genomes of interest were aligned using CLC SequenceViewer (www.cicbio.com). Primers were grouped into the most similarsequences. Mismatches were not allowed within the 10 most 3′ nucleotidesto have the best base-pairing directly upstream of where the polymerasecatalyzes incorporation of nucleotides. Once the sequences were aligned,they were grouped such that no more than two degenerate nucleotideswould be included in a single primer.

The sequences for the capture-specific probes were described in Jeney etal. except for slight modifications of HPV 6, 11, 16, and 18. Thecapture probe sequences are shown in FIG. 30 (Table 1, SEQ ID NOS:1-20). Probes were 5′ modified with a primary amine linked through a 6Carbon spacer arm.

All primers and probes were synthesized at Integrated DNA Technologies(Coralville, Iowa).

Design of Globin Primers and Probe

The human beta-globin gene was chosen as an internal positive controlnecessary to confirm successful nucleic acid purification from clinicalsamples. The forward and reverse primers were designed as follows:5′-GAA TAA CAG TGA TAA TTT CTG GG-3′ and 5′-GAA GAT AAG AGG TAT GAA CATGA-3′ (SEQ ID NO:21), respectively. The amino-terminated beta-globincapture probe was: 5′-ATC GAG CTG AAG GGC ATC GAC TTC AA-3′ (SEQ IDNO:22).

Polymerase Chain Reaction (PCR)

An extensive PCR optimization protocol was performed using plasmidscontaining full-length viral DNA for HPV 16 and HPV 18 based on theirprominence as high risk HPV subtypes. Preliminary experimentsdemonstrated that HPV 16 was more challenging to amplify than 18, andthus optimization was focused on HPV 16 amplification. Thermocyclingconditions similar to those described in Jeney et al. (Jeney C, TakacsT, Sebe A, Schaff Z: Detection and typing of 46 genital humanpapillomaviruses by the L1F/L1R primer system based multiplex PCR andhybridization. J Virol Methods 2007, 140(1-2):32-42) were performed inwhich PCR was performed for 10 cycles annealing at a lower temperature,and the for the remaining 25-35 cycles, annealing is performed at ahigher temperature. PCR was performed using 1000 copies of HPV 16containing plasmid over a background of 32 ng C33A purified nucleic acidand optimized for MgCl2 concentration, primer concentration, bufferconstituents, and annealing temperatures for each of the two annealingtemperatures. PCR was ultimately optimized under the followingconditions: 10 mM Tris-HCl, pH 9, 50 mM KCl, 100 μg/ml BSA, 1.5 mMMgCl2, 0.2 mM dATP, 0.2 mM dGTP, 0.2 mM dCTP, 0.075 mM dTTP, 0.125 mMdUTP, 0.2 μM of each individual primer, 0.002 units/μl heat stableuracil-DNA glycosylase, UDG, (USB Corp., Cleveland, Ohio) and 0.05units/μl GoTaq Hot Start Polymerase (Promega Corp., Madison Wis.).Thermocycling conditions were as follows: 37° C. 10 min (UDGactivation), 95° C., 2 min, 10 cycles of 95° C. 30 sec, 46° C. 30 sec,72° 30 sec, 25-35 cycles of 95° C. 30 sec, 49° C. 30 sec, 72° 30 sec,and a final extension at 72° C. for 3 min. These same conditions wereverified useful for beta-globin amplification. PCR optimization wasperformed on an MJ Mini Gradient Thermal Cycler (Biorad, Hercules,Calif.) and further confirmed on a Multigene II Thermal Cycler (Labnet,Woodbridge, N.J.).

Electrophoresis and Image Analysis

Following amplification, 2 μl 6× dye was added to 10 μl for a finalvolume of 12 μl. Samples were analyzed on 3% agarose gels melted in0.5×TAE (50×TAE is 2 M Tris acetate, 100 mM EDTA; 0.5×TAE is 20 mM Trisacetate, 1 mM EDTA) buffer to which 1/10,000 volume of GelGreen(Biotium, Hayward, Calif.) was added. Samples were electrophoresed for30-60 min at 100 Volts using a VWR mini Electrophoresis System (VWR,West Chester, Pa.). Wells were generated with 17 lane (4 mm wide) or 24lane (3 mm wide) combs to which 5 μl or 3 μl, respectively, of samplescontaining dye were loaded.

Gel bands were illuminated using a Dark Reader Transilluminator (ClareChemical Research, Delores, Colo.) and images were captured using a SonyCyber-shot DSCH2 Digital camera, ISO set to 80, F=3.5, shutter speed=3sec and timer=2 sec. Bands were quantitated using ImageJ software(http://rsb.info.nih.gov/ij/). Background was subtracted using a rollingball radius set to 50 pixels and the area under the peaks measuring thebands was collected.

Cloning of L1 Fragment from Clinical Samples

The HPV L1 amplified regions were cloned for specific types from DNApurified from clinical samples. To do this, specific HPV types wereidentified via amplification and reverse dot blot hybridization. HPV L1regions were cloned from samples containing single infections. Forwardand reverse primers were designed with type specific L1 sequences (seeFIG. 31, Table 2 (SEQ ID NOS: 23-46) flanked with Bam H1 and EcoR1restriction sites, respectively. The digested fragment was subsequentlygel purified and ligated into Bam H1/Eco R1 digested and gel purifiedpBS II KS+ (Bluescript) cloning vector. Successful amplification of theHPV insert was confirmed using the HPV primer mix, and specific type wasconfirmed via RDB and sequencing using an Applied Biosystems Automated3730 DNA Analyzer with Big Dye Terminator chemistry and Ampli-Taq-FS DNAPolymerase (Applied Biosystems, Inc., Foster City, Calif.).

Design of Spotting Control and Positive Control

A spotting control for the RDB filters was designed as follows:/5AmMC6/AAA AAA AAA AAA AAA AAA/3Bio/ (SEQ ID NO:47).

A positive control was designed that contained the forward primersequence to HPV 6/11, and the reverse primer sequence to HPV 42,flanking a non-HPV related sequence derived from the green fluorescentprotein, plasmid, pEGFP-C2. To do this, primers were designed with BamH1 and EcoR1 restriction sites flanking the forward and reverse primersequences, respectively, which in turn flanked GFP specific sequencesallowing for the amplification of a 258 bp GFP insert. The resultingforward and reverse primer sequences were 5′-GCT TGG ATC CCG TAA ACG TATTCC CTT ATT TTT TTA AAC GGC CAC AAG TTC AGC GTG-3′ (SEQ ID NO:48) and5′-AAG CGA ATT CAC TCT AAA TAC TCT GTA CTG TCT TGT AGT TGC CGT CGT CCTTGA-3′ (SEQ ID NO:49), respectively. Following amplification, the PCRproduct was purified and restricted with Bam H1 and Eco R1. The digestedfragment was subsequently gel purified and ligated into Bam H1/Eco R1digested and gel purified pBS II KS+(Bluescript) cloning vector.Successful amplification of the GFP fragment was confirmed using the HPVprimer mix.

Reverse Dot Blot (RDB)

Membrane filters were prepared following the method described in Zhanget al. (Zhang Y, Coyne M Y, Will S G, Levenson C H, Kawasaki E S:Single-base mutational analysis of cancer and genetic diseases usingmembrane bound modified oligonucleotides. Nucleic Acids Res 1991,19(14):3929-3933). Briefly, negatively charged nylon, 0.45 um, Biodyne Cmembranes (Pall Corporation, city, state) were pre-wet in 0.1 N HClfollowed by activation in 10%N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) for15 min. Membranes were rinsed in water and air-dried. Amino-terminatednucleic acid probes were resuspended in 0.5 M Sodium Bicarbonatecontaining 0.1% Tween, and spotted on the membranes using a BioRoboticsBiogrid (Boston, Mass.). Membranes were air-dried and dessicated untiluse. Immediately prior to use, membranes were incubated with 0.1 N NaOHto quench any remaining un-bound activated sites.

Amplicons, 5 μl, were mixed with 50 μl hybridization buffer (3×SSPE,0.1% SDS, 25% formamide), heated at 95° C. for 5 minutes, andimmediately placed on ice to prevent re-annealing. All membranemanipulations were performed at room temperature with gentle agitation.Membranes were prehybridized in 250 μl hybridization buffer for 15 minfollowed by the addition of denatured amplicon and hybridization for 1-3h. Following hybridization, filters were washed in 1.5 ml 0.1% SDS,twice for 10-15 min. Membranes were incubated with a 1:500 dilution ofhorseradish peroxidase conjugated streptavidin (Thermo FisherScientific, Inc., Waltham, Mass.) in 1×SSPE/0.1% SDS for 30 min followedby 3 rinses for 10 min each in 0.5×SSPE/0.3% SDS. One-Step TMB-BlottingSolution (Thermo Fisher Scientific, Inc.), 750 μl, was added to themembranes and color development performed for 10 min. Membranes werewashed for 10 min with 5 ml of water. Developed filters were scannedusing a Hewlett Packard Scanjet 4850 and/or photographed fordocumentation.

Results

Design of PCR Primers

FIG. 32 (Table 3A, SEQ ID NOS:50-77) and FIG. 33 (Table 3B, SEQ IDNOS:78-105) show the primers for amplifying the HPV L1 gene that weredesigned following the rules described in Materials and Methods.Following alignment starting at the 3′ ends, not allowing any mismatcheswithin the 10 most 3′ nucleotides, and using a maximum of 2 degeneratenucleotides per primer, a total of 8 forward and 8 reverse primersequences were designed. This reduces by half the number of individualprimers synthesized by Jeney et al. (Jeney C, Takacs T, Sebe A, SchaffZ: Detection and typing of 46 genital human papillomaviruses by theL1F/L1R primer system based multiplex PCR and hybridization. J VirolMethods 2007, 140(1-2):32-42) and thus reduces the overall costs of thePCR assay. In total, there were 13 forward and 21 reverse primersproduced from these degenerate sequences. Following this strategy, themix of primers contained within it an exact match for 16 of the 20 HPVtargets including all but one of the high risk types. Furthermore,although attempts were made to cover as much of the primer sequences aspossible, mismatches were not as much of a concern in the low risktypes. It was reasoned that although it is expected that all therelevant types will be amplified with these primers, in the unlikelyevent that a low risk is not amplified, a false negative under theseconditions is not as critical as a false negative for a high risk.Mismatches in the forward primer set included single mismatches for lowrisk 42 and 43, and 3 mismatches for low risk 44. Mismatches in thereverse primer set included single mismatches for low risk 11 and 44,and high risk 66. All mismatches, except one in the HPV 44 forwardsequence were within the 5′ end of the primer.

Validation of HPV Amplification

The HPV L1 primer set was initially tested on chimeric plasmidscontaining full-length or partial HPV sequences. FIG. 34 shows theresults of amplification of plasmid DNA for the various HPV types.Reaction mixtures containing 100 copies/μl of either full-length DNAsequence containing plasmids for HPV 6, 11, 16, 18, 52, or partialsequence for HPV 35 were subjected to 40 cycles of amplification. In allcases, HPV was amplified in the presence of 1.6 ng/μl C33A nucleicacids. All plasmids amplified with the primer set. There weredifferences among the plasmids with respect to how well each behaved asa template, with HPV 18 being a markedly better template than theothers.

To demonstrate more clearly the differences between the behaviors ofeach plasmid as a template for the L1 primer set, amplification wasperformed on two-fold serially diluted plasmid DNA. FIGS. 35A-D showresults of representative experiments in which HPV plasmid DNA wasamplified at 12 to 12,500 copies/μl of DNA over a background of 1.6ng/μl genomic nucleic acid per reaction. The upper images are of thegels used to separate the products of amplification of HPV 16 and HPV 18containing plasmids, and similar to what is seen in FIG. 34, HPV 18demonstrates better amplification at lower copy number than HPV 16. Thedata resulting from image analysis of these gels as well as similar gelsanalyzing products of amplification for the remaining plasmids are shownin FIG. 35B. The data from these graphs confirm the markedly betteramplification of HPV 18 compared to the other plasmids and similarlythat HPV 16 was less sensitive to amplification under these conditions.Amplification of HPV 18 began to plateau at approximately 200 copies/μl,amplification of HPV 11, 35 and 52 began to plateau at approximately 800copies/μl and amplification of HPV 16, the weakest template, began toplateau at approximately 1500 copies/μl.

In addition to the full-length and partial HPV clones, L1 target regionscloned directly from clinical samples were also subjected toamplification with the L1 primers. The graph in FIG. 35D shows theresults of image analysis of PCR of 10 copies/μl through 10,000copies/μl of 12 type-specific HPV clones. Similar to what was seen withthe full-length plasmids, some templates were more sensitive thanothers. Amplification of the most sensitive templates, HPV 18, 31, 33,51, 56, and 66 began to plateau at <150 copies/μl. Amplification of themore challenging templates began to plateau at >300 copies/μl with 16being the most challenging at 1500 copies/μl. The remaining templatesshow amplification plateaus between these lower and upper ranges. Takentogether, these data demonstrate that all targets of HPV tested weresuitable templates for amplification with the L1 primer set.

Preliminary experiments (data not shown) demonstrated that HPV andbeta-globin could not be amplified simultaneously in the same reactionmix without adversely affecting one another. Typically, if one templatewas in significant excess of the other template, competition for PCRreactants would occur making this a poor assay design for the ultimateproduct. Therefore, an assay was designed such that beta-globin and HPVcould be amplified in separate chambers, but would need to follow thesame thermal cycling conditions. For the experiments shown in FIGS. 35Aand 35B, the same samples were subjected to amplification withbeta-globin primers. The lower image in FIG. 35A shows a representativegel of globin amplified products. The samples for these experimentscontained the same input HPV and genomic DNA as with the HPVamplification curves. HPV input was increased whereas the genomic inputnucleic acid stayed constant at 1.6 ng/μl. As can be seen in the image,the level of input HPV had little or no effect on globin amplification.FIG. 35D shows the results of image analysis of the companion globingels run for each of the plasmid HPVs shown in FIG. 35B. These datademonstrate that regardless of the input of HPV, beta globinamplification remains relatively constant. Thus, whereasco-amplification of HPV and globin results in competition, the presenceof either template without the specific primers did not adversely affectamplification of the specific target.

Globin Curve

The internal control beta-globin was amplified in a separate chamberfrom HPV, but was subjected to the same temperature and cyclingconditions as HPV. Therefore, it was important to establish howbeta-globin amplification would behave under these conditions. Toaddress this question, serially diluted nucleic acid purified from C33Acells was subjected to amplification with beta-globin primers.Amplification was performed on 0.025-1.6 ng/μl of purified nucleic acidfor 30, 35, or 40 cycles. The results from these experiments are shownin FIG. 36. When PCR was performed for 40 cycles, amplicon productionwas completely saturated at <0.2 ng/μl input nucleic acid. In contrast,when performed for 30 or 35 cycles, amplification of globin began toplateau at 0.4 or 0.1 ng/μl, respectively, and did not saturateuntil >1.6 or >0.8 ng/μl, respectively. These data were generated onvarious days using C33A nucleic acid purified at different times,demonstrating the highly reproducible nature of this assay.

In the experiments in FIG. 36, template was presented in the context oftotal nucleic acid purified form C33 A cells, rather than purified DNA.Because the ultimate goal of these experiments was to design apoint-of-care microfluidic molecular diagnostic tool for HPV detection,each step of the assay was designed with manufacture of the end-productin mind. The use of ribonuclease treated nucleic acid resulting inRNA-free purified DNA was therefore considered. In experiments,ribonuclease in the purification module was determined unnecessary andpossibly detrimental to the actual recovery of nucleic acids from lowcell concentrations. That is, under conditions of limiting DNA, RNA mayact as a carrier to aid purification. Several purification experimentswere performed comparing the yields plus/minus ribonuclease treatment,and in the case of C33A cells, yields of nucleic acid were generally4-fold higher in the absence of ribonuclease treatment, suggesting thatthe actual amount of genomic DNA is approximately one quarter the actualyields determined via absorbance at 260 nm.

RDB HPV/Globin

Following PCR amplification of genomic and HPV DNA in separate vessels,the amplicons were combined and subjected to RDB on a single membranefilter. FIG. 37A shows the results of amplification of 10 copies/μl ofHPV over a background of 1.6 ng/μl genomic nucleic acid. The upper imageshows the gel image of HPV amplicons, and the bottom image shows thecorresponding globin amplicons. The relative intensities indicating thebehavior of each clone as a template is consistent with what was seenpreviously for 10 copies/μl (FIG. 37B and data not shown). FIG. 37Bshows the results of RDB of each combination of HPV and globin amplicon.Each clone was captured by its specific membrane bound probe andgenerated an unequivocal spot. In all but one instance the RDB spot wasstrong regardless of the intensity of the amplicon image. On the otherhand, the intensity of HPV 53 capture appeared to mimic low intensityamplification, but could still be identified.

Clinical Samples-Determination of Cell Numbers

Having established robust and reproducible conditions for HPV and globinamplification followed by specific detection on RDB, the assay wasvalidated using bona fide biological specimens. 117 clinical sampleswere obtained, subjected to nucleic acid purification, amplification ofboth HPV and globin, and final detection via RDB. All 117 samples werecollected and stored in storage transport medium (STM, Qiagen). Aportion of the samples were also treated with alkali denaturationreagent (Qiagen) prior to storage. Samples were frozen and stored forseveral weeks prior to nucleic acid purification. On average, thenucleic acids purified from samples stored with the addition ofdenaturant reagent gave slightly higher yields and purity when comparedto samples stored in STM alone. The average yield from thedenaturant/STM containing solutions versus STM alone was 11 and 4.6ng/μl respectively and the ratio of absorbance at 260/280 nm was 1.8 and1.6, respectively. Initially, 0.5 μl of each of these samples wereanalyzed for the presence of globin via PCR for 30 cycles, and eachsample, regardless of the concentration of nucleic acid generated, adetectable amplicon band measurable by pixel intensity and confirmed viaRDB detection. As seen in FIG. 38, although not directly correlatedunder these conditions, the trend of increasing pixel intensity followedthe trend of increasing ng/μl yields. Thus even in the absence ofdetectable nucleic acids (via absorbance, Nanodrop limitation ˜1 ng/μl),the assay produced a detectable globin amplicon following 30 cycles ofPCR, and thereby validated the sensitivity and usefulness of the assayfor clinical development.

Semi-quantitative PCR amplification of globin was used to calculate thenumber of genomes in the clinical samples. Reports suggest that avaginal swab contains ˜1-5 million cells (Depuydt C E, Benoy I H,Bailleul E J, Vandepitte J, Vereecken A J, Bogers J J: Improvedendocervical sampling and HPV viral load detection by Cervex-BrushCombi. Cytopathology 2006, 17(6):374-381; Quint W G, Pagliusi S R, LelieN, de Villiers E M, Wheeler CM: Results of the first World HealthOrganization international collaborative study of detection of humanpapillomavirus DNA. J Clin Microbiol 2006, 44(2):571-579; SchellenbergJ, Blake Ball T, Lane M, Cheang M, Plummer F: Flow cytometricquantification of bacteria in vaginal swab samples self-collected byadolescents attending a gynecology clinic. J Microbiol Methods 2008,73(3):216-226) equivalent to 6.4×10-6-3.2×10-5 g of pure DNA. UsingRNA-free DNA from C33A cells, and performing 30 cycles of PCR, a curveof pixel intensity versus ng input was generated (FIG. 39A). This wassimilar to the curve generated in FIG. 36, however, in this case thetotal input nucleic acid was genomic DNA and therefore directlycorrelated to the number of genomes.

To convert from ng/μl to # genomes the following equation was used:((ng/μl)/1E9)/3.2E-12 g/genome

To convert genomes to cells, divide the results from the above equationby 2.

To determine the number of genomes present in the clinical samples,various low, medium, and high yielding samples (FIG. 38) were analyzedby dilutions such that the resulting intensity of the amplicons wouldfall within the linear range of the assay. The results in FIG. 39B showthat the calculated range for samples stored in STM alone ranged from1-40 ng/μl. These numbers were approximately 2-7 fold higher than theng/μl obtained from absorbance at 260 nm. This was not surprising, sinceas described above, total nucleic acid purified in the absence ofribonuclease was, on average, 4-times greater than DNA purifiedfollowing ribonuclease treatment. Using the equation above, this rangecorresponded to 312.5-12,500 genomes/μl, or 156.25-6,250 cell/W. Theoriginal volume of these samples was 1 ml. Therefore, the number ofcells found in these samples ranged from 156,250-6,250,000.

FIG. 39C shows the calculated ng/μl for samples that were stored in STMand denaturant. Under these conditions, the difference betweencalculated ng/μl and ng/μl obtained from absorbance at 260 nm was not asgreat consistent with the hydrolysis of RNA in the presence of alkali.The calculated DNA range under these conditions was between 0.6-20ng/μl. This corresponded to 187.5-6250 genomes/μl or 93.75-3125 cells/W.The original volume of these samples was 1.5 ml. Therefore, the numberof cells found in these samples ranged from 140,625-4,687,500.

Taken together, these data demonstrate that using the semi-quantitativesystem, the number of cells from samples stored under differentconditions could be estimated, and that regardless of the storage, therange of cells calculated was consistent with previous reports. Thesedata provided a lower (150,000 cells) and higher (5,000,000 cells) limiton what to expect in vaginal samples and demonstrated that the assay wascompatible with the full range expected in the samples.

Clinical Samples and Demonstration of HPV

To demonstrate that the system could detect and distinguish HPV as wellas, or better than, the current accepted method of HPV detection, PCRwas performed on nucleic acid purified from all 117 clinical samplesusing the L1 primer set followed by RDB of the resulting amplicons.

FIG. 40 (Table 4) is the summary of results of PCR and RDB of theclinical samples. As shown in the table, 75% (88/117) of the samplesdemonstrated an HPV amplified band following PCR and 45% (40/88) of theHPV amplicon positives were captured by an HPV-specific probe on RDB.The most likely explanation for non-detection of the remaining wasabsence of specific capture probes for the HPV types. Of the HPV RDBpositive spots, 31/40 contained a high risk HPV type, present either asa single infection (21) multiple high risk infection (5) or combined lowand high risk infection (5). The remaining 9 RDB positive samples weredue to low risk single (7) or multiple (2) infections.

It was determined how well the assay compared with Digene HC2 (Qiagen,Valencia, Calif.) data in detecting high risk HPV. The data in FIG. 41Ashow the comparison of samples positive for HPV high risk using theDigene system versus the HPV result obtained with the system describedin this example. The assay confirmed high risk infectivity in all but 1of the Digene high risk samples. This sample (data not shown) resultedin Digene positive of 1.01, just barely considered Digene-positive forhigh risk. However, in multiple repeats of this sample, an HPV ampliconwas never detected. This suggests that this would have been read as afalse positive, albeit a very low positive. On the other hand, the assaysystem demonstrated in this example picked up several other high risksamples that were not detected using the Digene HC2 kit. As can be seenin FIG. 41B, the present assay system picked up 6 high risk samples thatwere read as negative by the Digene system, demonstrating thesuperiority sensitivity and specificity of the assay demonstrated inthis example.

Discussion

This example demonstrates the development of a HPV molecular diagnostictest that detects and distinguishes individual low and high risk HPVsubtypes. The method was developed for use on a fully automatedmicrofluidic (CARD) platform that required only the input of abiological specimen on the part of the user. The automated protocolreleases nucleic acids from the sample, subjects them to amplificationwith globin and HPV specific primers, and identifies specific subtypesof HPV via a reverse dot blot hybridization and detection. The exampledemonstrates the ability of the HPV assay to identify 20 differentinfectious HPV types as well as beta-globin from human epithelial cells.In addition, both the sensitivity of this assay as well as itsreproducibility was been demonstrated.

Preliminary studies (not shown) were aimed at testing previously knownPCR-based methods for the identification of HPV specific subtypes inbiological samples and determining if any of these methods were suitablefor use with the HPV assay, and/or if any could be improved upon.Criteria for a good target selection to be used on the CARD included:

1. The amplification of a short amplicon that would not requiresubstantial elongation time during PCR and thus result in decreasedoverall time for PCR.

2. The targeting of a hypervariable region to design specific captureprobes for all targets of interest and that could be expanded to includenew HPV subtypes as they become relevant.

3. Significant homology in the flanking regions of the target site tominimize the number of primers required to amplify all HPV targets ofinterest

Although many PCR-based methods known in the art rely on theamplification of a distinct nucleic acid sequence within the L1 gene,there do exist methods that rely on alternative genes within the HPVgenome (Josefsson A, Livak K, Gyllensten U: Detection and quantitationof human papillomavirus by using the fluorescent 5′ exonuclease assay. JClin Microbiol 1999, 37(3):490-496). FIG. 42 (SEQ ID NO:106) shows thesequence of the L1 gene of HPV 16 containing highlighted sequences (SEQID NOS:107-112) corresponding to the various target regions foramplification. SEQ ID NO:107 and SEQ ID NO:108 are L1 forward andreverse primers, respectively; SEQ ID NO:109 and SEQ ID NO:110 are SPFprimers; SEQ ID NO:110 and SEQ ID NO:111 are GP5/GP6; SEQ ID NO:109 andSEQ ID NO:112 are PGMY11/09.

Three of the established systems amplified all or part of a 450 bpregion residing within the middle of the L1 gene. The MY09/MY11 systemwas originally described in the early 1990s as a set of degenerativeprimers that could detect multiple HPV types (Hildesheim A, Schiffman MH, Gravitt P E, Glass A G, Greer C E, Zhang T, Scott D R, Rush B B,Lawler P, Sherman M E et al: Persistence of type-specific humanpapillomavirus infection among cytologically normal women. J Infect Dis1994, 169(2):235-240) resulting in an amplicon of approximately 450 bp.To avoid some of the problems that may arise with the use ofdegenerative primers, Gravitt et al. (Gravitt P E, Peyton C L, Alessi TQ, Wheeler C M, Coutlee F, Hildesheim A, Schiffman M H, Scott D R, AppleR J: Improved amplification of genital human papillomaviruses. J ClinMicrobiol 2000, 38(1):357-361) modified the MY09/MY11 system anddesigned a pool of 5 forward and 13 reverse primers. Individualcombinations of these primers would allow amplification of any of thetarget HPV types. Targeting the same region, Snijders et al. (Snijders PJ, van den Brule A J, Schrijnemakers H F, Snow G, Meijer C J, WalboomersJ M: The use of general primers in the polymerase chain reaction permitsthe detection of a broad spectrum of human papillomavirus genotypes. JGen Virol 1990, 71 (Pt 1):173-181) identified single sequence primerswhich were further modified by elongation at the 3′ ends (de Roda HusmanA M, Walboomers J M, van den Brule A J, Meijer C J, Snijders P J: Theuse of general primers GP5 and GP6 elongated at their 3′ ends withadjacent highly conserved sequences improves human papillomavirusdetection by PCR. J Gen Virol 1995, 76 (Pt 4):1057-1062) and expanded tocover a broader range of HPV types (Schmitt M, Dondog B, Waterboer T,Pawlita M: Homogeneous amplification of genital human alphapapillomaviruses by PCR using novel broad-spectrum GP5+ and GP6+primers. J Clin Microbiol 2008, 46(3):1050-1059). The size of theamplicon generated by this “general primer” system was 150 bp.

Targeting the same region of the L1 gene, Kleter and colleagues (KleterB, van Doorn L J, Schrauwen L, Molijn A, Sastrowijoto S, ter Schegget J,Lindeman J, ter Harmsel B, Burger M, Quint W: Development and clinicalevaluation of a highly sensitive PCR-reverse hybridization line probeassay for detection and identification of anogenital humanpapillomavirus. J Clin Microbiol 1999, 37(8):2508-2517; Kleter B, vanDoom U, ter Schegget J, Schrauwen L, van Krimpen K, Burger M, terHarmsel B, Quint W: Novel short-fragment PCR assay for highly sensitivebroad-spectrum detection of anogenital human papillomaviruses. Am JPathol 1998, 153(6):1731-1739) designed a system of 4 forward and 2reverse primers capable of recognizing all HPV types by theincorporation of the “universal base”, inosine. These primers amplifieda 65 bp region that included within the amplicon a hypervariable regionthat could be targeted directly for type-specific capture. Focusing onthe upstream L1 sequence, Yoshikawa, et al. (Yoshikawa H, Kawana T,Kitagawa K, Mizuno M, Yoshikura H, Iwamoto A: Detection and typing ofmultiple genital human papillomaviruses by DNA amplification withconsensus primers. Jpn J Cancer Res 1991, 82(5):524-531) describes theL1C1/L1C2 primers that were later modified to include up to 46 distincttypes of HPV (Jeney C, Takacs T, Sebe A, Schaff Z: Detection and typingof 46 genital human papillomaviruses by the L1F/L1R primer system basedmultiplex PCR and hybridization. J Virol Methods 2007, 140(1-2):32-42;Takacs T, Jeney C, Kovacs L, Mozes J, Benczik M, Sebe A: Molecularbeacon-based real-time PCR method for detection of 15 high-risk and 5low-risk HPV types. J Virol Methods 2008, 149(1):153-162). The studyundertaken by Jeney (Jeney C, Takacs T, Sebe A, Schaff Z: Detection andtyping of 46 genital human papillomaviruses by the L1F/L1R primer systembased multiplex PCR and hybridization. J Virol Methods 2007,140(1-2):32-42; Takacs T, Jeney C, Kovacs L, Mozes J, Benczik M, Sebe A:Molecular beacon-based real-time PCR method for detection of 15high-risk and 5 low-risk HPV types. J Virol Methods 2008,149(1):153-162) to identify novel regions that would allow for moresensitive detection of more types, identified a region within the HPV L1sequence that was consistent with one of the hypervariable regions ofthe proteins identified by X-ray crystallographic studies (Chen X S,Garcea R L, Goldberg I, Casini G, Harrison S C: Structure of smallvirus-like particles assembled from the L1 protein of humanpapillomavirus 16. Mol Cell 2000, 5(3):557-567) and further confirmed bydomain swapping experiments (Olcese V A, Chen Y, Schlegel R, Yuan H:Characterization of HPV16 L1 loop domains in the formation of atype-specific, conformational epitope. BMC Microbiol 2004, 4:29). Use ofthe primers described in Jeney C, Takacs T, Sebe A, Schaff Z (Detectionand typing of 46 genital human papillomaviruses by the L1F/L1R primersystem based multiplex PCR and hybridization. J Virol Methods 2007,140(1-2):32-42) and Takacs T, Jeney C, Kovacs L, Mozes J, Benczik M,Sebe A (Molecular beacon-based real-time PCR method for detection of 15high-risk and 5 low-risk HPV types. J Virol Methods 2008,149(1):153-162) results in a ˜250 bp amplicon.

Testing of each of these aforementioned L1 amplification methods wasperformed. Success was achieved with each system. Targeting the upstreamL1 250 bp region was chosen for further studies owing to its ampliconsize and hypervariability within the capture probe region, which allowedfor better design of capture probes that could be hybridized at roomtemperature.

As more becomes known regarding HPV infections and the potentialdevelopment of cervical cancer, it is becoming clear that in addition todemonstrating the presence of high risk HPV types, understanding theviral load, or number of HPV particles per cell, will also contribute todiagnosis. To examine this using the assay, the pixel intensity of HPVand globin from the same samples was evaluated. FIG. 43A shows theresults of plotting increasing HPV pixel intensity with thecorresponding globin pixel intensity. As shown in FIG. 43A, there is nocorrelation between globin pixel intensity and increasing HPV pixelintensity.

The ratio of HPV to globin pixels was then compared to the presence ofhigh risk HPV types. FIG. 43B shows the ratio of HPV to globin pixelsand was plotted versus low to high risk HPV types. These ratios variedfrom <1 to >6, but there did not appear to be any obvious correlationwith the presence of HPV types. FIG. 43C shows the ratio of HPV toglobin pixels plotted versus low to high risk HPV types. The “Digene”series plots the numbers which are the results of the commerciallyavailable Digene assay in comparison to with the present assay. Theinset chart shows the Digene assay results (with a different scaleY-axis) that were then scaled and incorporated in the main graph.

In summary, the HPV assay conducted on the CARD provides a fullyautomated system for the rapid and reliable molecular detection ofclinically relevant HPV types. Furthermore, owing to the portability ofthe CARD, the methods demonstrated in this example have widespreadapplication in both industrialized and developing nations.

Example 5 CARD-Based Detection of a Sparse Target

This example demonstrates the detection of a sparse target. In thisexample, the sparse target nucleic acid is associated with a waterbornepathogen. Detection of the sparse target nucleic acid is indicative ofits presence in a water supply. Such an assay can be used to detectsparse targets (from small numbers of organisms or cell nuclei) in alarge liquid sample volume.

In reference to FIGS. 20A-20P the following operations and reagents wereused to perform an assay for a live waterborne pathogen, Cryptosporidiumparvum.

Sample Addition and Immunomagnetic Separation

All reagents are commercially available from Dynal for immunomagneticcapture of Cryptosporidium parvum. Volumes were adjusted for automatedoperation on the CARD.

a. An operator adds 1 ml of a pre-concentrated water sample into thesample input reservoir.

b. Dispense 100 μl of buffer 1, 100 □l of buffer 2 and 10 μl of beadsinto the same tube as the sample. Incubate for 30 minutes with gentleagitation or fluffing.

c. Raise the magnet under the sample input reservoir and pump thecontents of the reservoir to waste.

d. Dispense 200 μl of buffer 1 into the buffer 1 reservoir and afterlowering the magnet pump it to the sample input reservoir to re-suspendthe beads.

e. Raise the magnet under the sample input reservoir and pump thecontents of the reservoir to waste.

Heat Shock

Heat shock is optionally carried out to test for live organisms capturedin the immunomagnetic separation step described above. (Otherwise,proceed to DNA amplification with standard PCR methods as detailedbelow.)

Amplification is conducted using RNA amplification reagents and an RNAamplification technique (e.g., RT-PCR or NASBA or art-knownequivalents).

a. Lower the magnet.

b. Dispense 40 μl of nuclease free water into the non-heated magneticseparation reservoir and pump the entire contents into the heatedmagnetic separation reservoir and gently fluff to re-suspend the beads.

c. Turn on the heater to 42° C. for 5 minutes. Living Crypto will beginto express an RNA coding for a heat shock protein.

Lysis and Purification

All reagents are commercially available from Qiagen. Volumes wereadjusted for automated operation on the CARD.

a. Dispense 100 μl of Qiagen lysis buffer RLT into the lysis bufferreservoir and pump it to the sample input reservoir and agitate byfluffing 6 times. Then increase the temperature of the heater to 60° C.and incubate for 10 minutes. Fluff twice during the 10 minutes once at 5minutes and then just prior to 10 minutes.b. Dispense 100 μl of ethanol into the ethanol reservoir and pump it tothe sample input reservoir and fluff 6 times.c. Pump the entire contents of the sample input reservoir to the top ofthe silica filter and then pull the contents through the filter and pumpit to waste. Pull air through the filter by opening the valves connectedto the vacuum port and applying a vacuum for 30 seconds.d. Dispense 100 μl of ethanol into the ethanol reservoir and pump it tothe top of the silica filter and then pull the contents through thefilter and pump it to waste. Pull air through the filter by opening thevalves connected to the vacuum port and applying a vacuum for 30seconds.e. Dispense 100 μl of buffer 2 into the buffer 2 reservoir and pump itto the top of the silica filter and then pull the contents through thefilter and pump it to waste. Pull air through the filter by opening thevalves connected to the vacuum port and applying a vacuum for 30seconds.f. Dispense 100 μl of buffer 3 into the buffer 3 reservoir and pump itto the top of the silica filter and then pull the contents through thefilter and pump it to waste. Pull air through the filter by opening thevalves connected to the vacuum port and applying a vacuum for 30 secondsand repeat once.g. Dispense 100 μl of ethanol into the ethanol reservoir and pump it tothe top of the silica filter and then pull the contents through thefilter and pump it to waste. Pull air through the filter by opening thevalves connected to the vacuum port and applying a vacuum for 30seconds.h. Dispense 40 μl of nuclease free water into the non-heated magneticseparation reservoir and pump the entire contents up through the bottomof the silica filter and incubate for 2 minutes then pump the entirecontents back to the non-heated magnetic separation reservoir.

mRNA Separation

All reagents are commercially available from Dynal volumes were adjustedfor automated operation on the CARD.

a. Dispense 100 μl of binding buffer and then dispense 20 □l of beadsinto the non-heated magnetic separation reservoir then pump the bindingbuffer into the non-heated magnetic separation reservoir. Incubate for 5minutes with gentle fluffing.

b. Raise magnet under the non-heated magnetic separation reservoir andpump the contents to waste.

c. Dispense 100 μl of wash buffer A into the wash buffer A reservoir andpump it into the non-heated magnetic separation reservoir. Lower themagnet and re-suspend the beads with gentle fluffing.

d. Raise magnet under the non-heated magnetic separation reservoir andpump the contents to waste.

e. Repeat steps c and d again.

f. Dispense 100 μl of wash buffer B into the wash buffer B reservoir andpump it into the non-heated magnetic separation reservoir. Lower themagnet and re-suspend the beads with gentle fluffing.

g. Raise magnet under the non-heated magnetic separation reservoir andpump the contents to waste.

h. Repeat steps f and g again.

i. While the non-heated magnetic separation reservoir is empty (exceptfor the beads) back pump air to the wash buffer B reservoir to clear thechannels and diaphragms of any residual fluids.

NASBA Amplification

a. Lower the magnet under the non-heated magnetic separation reservoir.

b. Dispense 30 μl of NASBA master mix amplification master mix reservoirand pump it to the non-heated magnetic separation reservoir. Fluff thecontents of the non-heated magnetic separation reservoir to re-suspendthe beads.

c. Pump the contents of the non-heated magnetic separation reservoirback into the amplification master mix reservoir

d. Increase the temperature of the multipurpose heater to 41° C.

e. Pull the contents of the amplification master mix reservoir throughthe amplification reactor and pump a small amount into waste. Thisprocess completely fills the amplification reactor with fluid (no airbubbles).

f. Incubate the contents of the amplification reactor for 90 minutes at41° C.

Lateral Flow Analysis

a. Dispense 20 μl of hybridization buffer into the hybridization bufferreservoir and pump an initial 4 μl portion of it to waste to clear thechannel and the diaphragm.

b. Pump 4 μl to the hybridization buffer to the heated analysisreservoir.

c. Pump an initial volume of 14 μl from the amplification reactor towaste to clear the channel and the diaphragm.

d. Then pump 2 μl from the amplification reactor into the heatedanalysis reservoir and then 6 μl from the hybridization buffer reservoirinto the heated analysis reservoir.

e. Dispense 4 μl of marker (in this case liposomes tagged with anoligonucleotide homologous to the amplicons and filled with a markerthat will be visible at the end point of the lateral flow analysis) intothe heated analysis reservoir. Incubate with gentle agitation at 41° C.for 5 minutes.f. Pump 8 μl of the contents of the heated analysis reservoir to thelateral flow strip in the analysis reservoir.g. Concurrently dispense 35 μl of running buffer into the running bufferreservoir and pump it to the lateral flow strip after the initial volumeof 8 μl or solution from the heated analysis reservoir is pumped to thelateral flow strip.h. The tagged liposomes if they hybridized with the amplicons will becaptured by another complimentary oligonucleotide attached to thelateral flow test strip and the internal die contained in the lipsomewill be visible at the same point.

Image Analysis

a. Position the camera over the analysis membrane and record the image.

b. Send the image to the control system for processing.

c. Report the results.

FIG. 44 shows results obtained from the CARD-based detection. Heat shockmRNA from Cryptosporidium parvum was detected. IMS=immunomagneticseparation. HS=heat shock.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. The term “connected” is to beconstrued as partly or wholly contained within, attached to, or joinedtogether, even if there is something intervening.

The recitation of ranges of values herein are merely intended to serveas a shorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminateembodiments of the invention and does not impose a limitation on thescope of the invention unless otherwise claimed.

No language in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the present inventionwithout departing from the spirit and scope of the invention. There isno intention to limit the invention to the specific form or formsdisclosed, but on the contrary, the intention is to cover allmodifications, alternative constructions, and equivalents falling withinthe spirit and scope of the invention, as defined in the appendedclaims. Thus, it is intended that the present invention cover themodifications and variations of this invention provided they come withinthe scope of the appended claims and their equivalents.

We claim:
 1. A self-contained, biological assay apparatus, comprising: ahousing; a dispensing platform including a controllably-movable reagentdispensing system, disposed in the housing; a reagent supply componentdisposed in the housing; a pneumatic manifold removeably disposed in thehousing in a space shared by the dispensing platform, removeably coupledto a microfluidic system having a fluidic transport layer and aplurality of reservoirs which are fluidly connected to said fluidictransport layer, wherein the fluidic transport layer, the reservoirs,and a test sample to be introduced into the fluidic transport layer aredisposed in the housing in the space separate from the dispensingplatform; a pneumatic supply system removeably coupled to the pneumaticmanifold in the housing in a space separate from the dispensingplatform; and a control system coupled to at least one of the dispensingplatform and the pneumatic supply system, disposed in the housing,wherein the apparatus is characterized as a self-contained, fullyautomated biological assay-performing apparatus, further wherein thetest sample, once introduced, is transported from a starting location inthe fluidic transport layer to the analysis reservoir separately fromany other samples and separately from the pneumatic manifold and thedispensing system.
 2. The apparatus of claim 1, wherein the dispensingplatform further comprises: a motion control system operatively coupledto the reagent dispensing system, wherein the reagent dispensing systemincludes a reagent dispenser component having a distal dispensing end;and a camera connected to the reagent dispensing system having a fieldof view that includes at least a selected region of interest of thereservoirs.
 3. The apparatus of claim 2, wherein the reagent dispensercomponent is a needle structure having a length, L, and a selected boresize.
 4. The apparatus of claim 3, wherein the reagent dispensing systemfurther includes a reagent storage structure fluidly connected to thereagent dispenser component, wherein the reagent storage structure has aselected dimension that operatively coincides with the selected boresize.
 5. The apparatus of claim 4, wherein the reagent storage structureis a length of tubing.
 6. The apparatus of claim 2, wherein the reagentdispenser component is a plurality of needle structures of length, L,having different respective, selected bore sizes.
 7. The apparatus ofclaim 6, wherein the reagent dispensing system further includes aplurality of reagent storage structures that are respectively fluidlyconnected to the plurality of needle structures, wherein each reagentstorage structure has a selected dimension that operatively coincideswith the respective selected bore size.
 8. The apparatus of claim 7,wherein each of the reagent storage structures is a length of tubing. 9.The apparatus of claim 7, having only two reagent storage structuresthat are respectively fluidly connected to two reagent dispensercomponents, wherein one of the reagent dispenser components has a borediameter bi, 0.003<bi<0.018 inches and the other reagent dispensercomponent has a bore diameter b2, 0.015<b2<0.030 inches.
 10. Theapparatus of claim 1, wherein the pneumatic manifold is interfaced withthe microfluidic system.
 11. The apparatus of claim 10, wherein themicrofluidic system further comprises a multi-layer, monolithic,polymeric, non-elastomeric microfluidic component having a givenconfiguration of microfeatures including a plurality ofpneumatically-activated diaphragms.
 12. The apparatus of claim 11,wherein the pneumatic manifold has a plurality of pneumatic-only portson an underside thereof, and a plurality of pneumatic-only channelsdisposed therein in fluid connection with a plurality of valves in thefluidic transport layer and the plurality of pneumatic-only ports,wherein said plurality of pneumatic ports have a fixed configuration,and said plurality of pneumatic-only channels have a given configurationcorresponding to a given configuration of the plurality ofpneumatically-activated diaphragms in the fluidic transport layer. 13.The apparatus of claim 12, wherein the pneumatic supply system furthercomprises a plurality of aperture tubes that provide a passage of thepneumatic signal there through, in fluid connection with said pluralityof pneumatic-only ports, wherein said plurality of aperture tubes have afixed configuration corresponding to the fixed configuration of theplurality of pneumatic-only ports of the pneumatic manifold, removeablyconnected to the pneumatic manifold.
 14. The apparatus of claim 11,wherein the multi-layer, monolithic microfluidic component furthercomprises: a polymeric, non-elastomeric substrate having a plurality offluid channels disposed therein, each of the fluid channels having aninlet end and an outlet end; at least one reagent reservoir of a typecapable of holding a reagent material; at least one bi-directionaldiaphragm pump comprising at least three non-elastomeric membrane-baseddiaphragm valve structures; and a valve disposed in fluid coupling withthe at least one reagent reservoir and at least one of the inlet ends,wherein the valve is adapted to controllably direct a flow of thematerial from the at least one reagent reservoir to a plurality ofreservoirs via at least one of the channels coupled to the valve,further wherein the multi-layer, monolithic microfluidic componentconsists of a non-elastomeric, polymeric material.
 15. The apparatus ofclaim 14, wherein the substrate further comprises a plurality ofanalysis reservoirs, each analysis reservoir including an analysissystem disposed therein.
 16. The apparatus of claim 14, wherein theanalysis system is one of colorimetric, fluorescent colorimetric,chemiluminescent, electrochemical, electrophoretic, lateral flow,protein microarray, nucleic acid microarray, fluorescent.
 17. Theapparatus of claim 14, further comprising a securing-ring structurehaving a plurality of perimeter indentations, wherein the analysismembrane is operatively engaged with the securing-ring structure. 18.The apparatus of claim 17, wherein the reservoir has at least a partialinner perimetal shelf upon which the analysis membrane is disposed. 19.The apparatus of claim 17, wherein the securing-ring structure comprisestwo opposing ring structures each having a plurality of perimeterindentations, further wherein the analysis membrane is disposedintermediate the two opposing ring structures.
 20. The apparatus ofclaim 15, further comprising a heater.
 21. The apparatus of claim 20,further comprising a heatable, magnetically engageable reservoir,wherein the heater is disposed adjacent the heatable, magneticallyengageable reservoir in such a manner that the heatable, magneticallyengageable reservoir can be heated.
 22. The apparatus of claim 14,further comprising: a tube mounting layer attached to a bottom surfaceof the substrate including a plurality of tubes each having a proximalend that is fluidly connected to a respective fluidic channel and adistal end projecting downwardly perpendicularly from the substrate; anda respective plurality of amplification/reaction chambers attached at atop region thereof to the tube mounting layer such that the distal endof each tube is disposed substantially near a bottom region thereof. 23.The apparatus of claim 22, further comprising a heater for elevating atemperature in each of the amplification/reaction chambers.
 24. Theapparatus of claim 11, further comprising a magnetic assembly operablydisposed under a reservoir or channel, wherein the magnetic assemblyfurther comprises a magnet, a magnet holder, a piston rod, and apneumatic piston assembly.
 25. The apparatus of claim 24, furthercomprising a heater assembly operably connected to the magneticassembly.